Combination therapy employing a pd1-lag3 bispecific antibody and a cd20 t cell bispecific antibody

ABSTRACT

The invention relates to combination therapies employing anti-PD1/anti-LAG3 bispecific antibody and a CD20 T cell-activating bispecific antibody, the use of these combination therapies for the treatment of cancer and methods of using the combination therapies.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of International Application No.PCT/EP2022/050040, filed Jan. 4, 2022, which claims priority to EuropeanApplication No. 21150425.3 filed on Jan. 6, 2021, the contents of whichare hereby incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing with 103 sequencessubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created Jul. 3, 2023, is namedP36645-US_Sequence_Listing.xml and is 137,621 bytes in size.

FIELD OF THE INVENTION

The present invention relates to combination therapies employing aPD1-LAG3 bispecific antibody and a CD20 T cell-activating bispecificantibody, the use of these combination therapies for the treatment ofcancer and methods of using the combination therapies.

BACKGROUND

B-cell proliferative disorders describe a heterogeneous group ofmalignancies that includes both leukemias and lymphomas. Lymphomasdevelop from lymphatic cells and include two main categories: Hodgkinlymphomas (HL) and the non-Hodgkin lymphomas (NHL). In the UnitedStates, lymphomas of B cell origin constitute approximately 80-85% ofall non-Hodgkin lymphoma cases, and there is considerable heterogeneitywithin the B-cell subset, based upon genotypic and phenotypic expressionpatterns in the B-cell of origin. For example, B cell lymphoma subsetsinclude the slow-growing indolent and incurable diseases, such asFollicular lymphoma (FL) or chronic lymphocytic leukemia (CLL), as wellas the more aggressive subtypes, mantle cell lymphoma (MCL) and diffuselarge B cell lymphoma (DLBCL). Despite the availability of variousagents for the treatment of B-cell proliferative disorders, there is anongoing need for development of safe and effective therapies to prolongremission and improve cure rates in patients.

An anti-CD20/anti-CD3 bispecific antibody is a molecule that targetsCD20 expressed on B cells and CD3 epsilon chain (CD3ε) present on Tcells. Simultaneous binding leads to T-cell activation and T-cellmediated killing of B cells. In the presence of CD20⁺ B cells, whethercirculating or in tissue, pharmacologically active doses of a CD20-CD3bispecific antibody will trigger T-cell activation and associatedcytokine release. Parallel to B cell depletion in the peripheral blood,CD20 T cell-activating bispecific antibody leads to a transient decreaseof T cells in the peripheral blood within 24 hours after the firstadministration and to a peak in cytokine release, followed by rapidT-cell recovery and return of cytokine levels to baseline within 72hours. Two major reported escape mechanisms during treatment with a Tcell-activating bispecific antibody include increased frequencies ofregulatory T cells (T_(regs)) and increased levels of PD-L1 expressionon B-precursor cells. T_(regs) suppress effector T cell activationthrough CTLA4 and other mechanisms. However, even when T cells are fullyactivated, upregulation of PD1 will lead to inhibitory signaling afterbinding to PD-L1 expressed by the tumor cells. These mechanisms induceeffector T cell suppression and exhaustion or dysfunction, which can betreated with checkpoint blockade.

Exhausted T cells are characterized by sustained expression of theinhibitory molecule PD-1 (programmed cell death protein 1) and it hasbeen found that blockade of PD-1 and PD-L1 (PD-1 ligand) interactionscan reverse T cell exhaustion and restore antigen-specific T cellresponses. However, targeting the PD-1-PD-L1 pathway alone does notalways result in reversal of T cell exhaustion, possibly due toresistance mechanisms, immunosuppressive activity of MDSC, and/orregulatory T cells.

Lymphocyte activation gene-3 (LAG3 or CD223) was initially discovered inan experiment designed to selectively isolate molecules expressed in anIL-2-dependent NK cell line (Triebel F et al., Cancer Lett. 235 (2006),147-153). LAG3 is a unique transmembrane protein with structuralhomology to CD4 with four extracellular immunoglobulin superfamily-likedomains (D1-D4). The membrane-distal IgG domain contains a short aminoacid sequence, the so-called extra loop that is not found in other IgGsuperfamily proteins. The intracellular domain contains a unique aminoacid sequence (KIEELE, SEQ ID NO:103) that is required for LAG3 to exerta negative effect on T cell function. LAG3 can be cleaved at theconnecting peptide (CP) by metalloproteases to generate a soluble form,which is detectable in serum. Like CD4, the LAG3 protein binds to MHCclass II molecules, however with a higher affinity and at a distinctsite from CD4 (Huard et al. Proc. Natl. Acad. Sci. USA 94 (1997),5744-5749). LAG3 is expressed by T cells, B cells, NK cells andplasmacytoid dendritic cells (pDCs) and is upregulated following T cellactivation. It modulates T cell function as well as T cell homeostasis.Subsets of conventional T cells that are anergic or display impairedfunctions express LAG3. LAG3⁺ T cells are enriched at tumor sites andduring chronic viral infections (Sierro et al Expert Opin. Ther. Targets15 (2011), 91-101). It has been shown that LAG3 plays a role in CD8 Tcell exhaustion (Blackburn et al. Nature Immunol. 10 (2009), 29-37).Thus, there is a need for antibodies that antagonize the activity ofLAG3 and can be used to generate and restore immune response to tumors.

By targeting both PD-1 and LAG-3 on dysfunctional tumour-specific Tlymphocytes, PD1-LAG3 aims to restore an effective anti-tumorimmune-response and to provide survival benefit to more cancer patientsthan the currently available checkpoint inhibitors. By preferentiallytargeting PD-1/LAG-3 co-expressing dysfunctional T cells and potentiallyreduced targeting of LAG-3 expressing Tregs in the tumormicroenvironment, PD1-LAG3 BsAb might avoid reinvigorating Treg mediatedimmunosuppressive effects while restoring the anti-tumor immuneresponse.

While effective CD20-expressing cancer therapies exist, suboptimalresponse, relapsed-refractory disease, and/or resistance to one or moretherapeutic agents have remained a challenge. Further, patients withhigher risk and cytogenetic abnormalities still have less than optimalresponse to approved therapies and shorter duration of response andprogression free survival. Accordingly, there is a need for moreeffective, safe, and durable targeted combination therapies for thetreatment of hematological malignancies.

SUMMARY OF THE INVENTION

The present invention relates to combination therapies employing ananti-CD20/anti-CD3 bispecific antibody and a bispecific antibodycomprising a first antigen binding domain that specifically binds toprogrammed cell death protein 1 (PD1) and a second antigen bindingdomain that specifically binds to Lymphocyte activation gene-3 (LAG3).It has been found, that the anti-PD1/anti-LAG3 bispecific antibodies asdescribed herein are advantageous over anti-PD1 antibodies as theyprovide better selectivity and efficacy. These anti-PD1/anti-LAG3bispecific antibodies are further characterized in that they show areduced sink effect (as shown by reduced internalization by T cells),they preferentially bind to conventional T cells as to Tregs and areable to rescue T cell effector functions from Treg suppression, theyshow increased tumor-specific T cell effector functions and increasedtumor eradication in vivo. Based on these properties they areadvantageous to be used in combination with T cell bispecificantibodies, in particular anti-CD20/anti-CD3 bispecific antibodies.

Described herein is an anti-CD20/anti-CD3 bispecific antibody for use ina method of treating cancer, in particular CD20 expressing cancer,wherein the anti-CD20/anti-CD3 bispecific antibody is used incombination with an anti-PD1/anti-LAG3 bispecific antibody.

The invention provides an anti-CD20/anti-CD3 bispecific antibody for usein a method as defined herein before, wherein the anti-PD1/anti-LAG3bispecific antibody comprises a first antigen binding domain thatspecifically binds to programmed cell death protein 1 (PD1) and a secondantigen binding domain that specifically binds to Lymphocyte activationgene-3 (LAG3), wherein a first antigen binding domain specificallybinding to PD1 comprises a VH domain comprising

-   -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1,    -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2,        and    -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID NO:3;        and        a VL domain comprising    -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:4;    -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5,        and    -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

In one aspect, provided is an anti-CD20/anti-CD3 bispecific antibody foruse in a method of treating CD20 expressing cancer, wherein theanti-CD20/anti-CD3 bispecific antibody and the anti-PD1/anti-LAG3bispecific antibody are administered together in a single composition oradministered separately in two or more different compositions.

Furthermore, provided is an anti-CD20/anti-CD3 bispecific antibody foruse in a method of treating CD20 expressing cancer, wherein theanti-CD20/anti-CD3 bispecific antibody is used in combination with ananti-PD1/anti-LAG3 bispecific antibody and wherein theanti-PD1/anti-LAG3 bispecific antibody comprises a Fc domain that is anIgG Fc domain, particularly an IgG1 Fc domain or an IgG4 Fc domain, andwherein the Fc domain comprises one or more amino acid substitution thatreduces binding to an Fc receptor, in particular towards Fcγ receptor.More particularly, the anti-PDT/anti-LAG3 bispecific antibody comprisesan Fc domain of human IgG1 subclass with the amino acid mutations L234A,L235A and P329G (numbering according to Kabat EU index).

In one aspect, provided is an anti-CD20/anti-CD3 bispecific antibody foruse in a method as described herein before, wherein theanti-PD1/anti-LAG3 bispecific antibody comprises a second antigenbinding domain that specifically binds to LAG3 comprising

-   -   (a) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:11,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:12, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:13; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:14,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:            15, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO: 16; or    -   (b) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:19,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:            20, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:21; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:22,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:23, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:24.

In another aspect, provided is an anti-CD20/anti-CD3 bispecific antibodyfor use in a method as disclosed herein, wherein the anti-PD1/anti-LAG3bispecific antibody comprises a first antigen-binding domainspecifically binding to PD1 comprising the VH domain comprising theamino acid sequence of SEQ ID NO: 9 and the VL domain comprising theamino acid sequence of SEQ ID NO:10.

In a further aspect, provided is an anti-CD20/anti-CD3 bispecificantibody for use as described herein, wherein the anti-PD1/anti-LAG3bispecific antibody comprises a second antigen-binding domainspecifically binding to LAG3 comprising

-   -   (a) a VH domain comprising the amino acid sequence of SEQ ID        NO:17 and a VL domain comprising the amino acid sequence of SEQ        ID NO:18, or    -   (b) a VH domain comprising the amino acid sequence of SEQ ID NO:        25 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 26.

In an additional aspect, provided is an anti-CD20/anti-CD3 bispecificantibody for use in a method as described herein, wherein theanti-PD1/anti-LAG3 bispecific antibody comprises a secondantigen-binding domain specifically binding to LAG3 comprising

-   -   (a) a VH domain comprising the amino acid sequence of SEQ ID NO:        27 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 28, or    -   (b) a VH domain comprising the amino acid sequence of SEQ ID NO:        29 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 30, or    -   (c) a VH domain comprising the amino acid sequence of SEQ ID NO:        31 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 32, or    -   (d) a VH domain comprising the amino acid sequence of SEQ ID NO:        33 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 34.

Furthermore, provided is an anti-CD20/anti-CD3 bispecific antibody foruse in a method as disclosed herein, wherein the anti-PD1/anti-LAG3bispecific antibody comprises

-   -   a first antigen binding domain specifically binding to PD1        comprising a VH domain comprising the amino acid sequence of SEQ        ID NO: 9 and a VL domain comprising the amino acid sequence of        SEQ ID NO: 10,    -   and a second antigen binding domain specifically binding to LAG3        comprising a VH domain comprising the amino acid sequence of SEQ        ID NO: 17 and a VL domain comprising the amino acid sequence of        SEQ ID NO: 18.

In a further aspect, provided is an anti-CD20/anti-CD3 bispecificantibody for use in a method of treating CD20 expressing cancer, whereinthe anti-PD1/anti-LAG3 bispecific antibody comprises a Fab fragmentspecifically binding to PD1 and a Fab fragment specifically binding toLAG3. In one aspect, the anti-PD1/anti-LAG3 bispecific antibodycomprises a Fab fragment specifically binding to PD1, wherein thevariable domains VL and VH are replaced by each other so that VL is partof the heavy chain and VH is part of the light chain.

In another aspect, provided is an anti-CD20/anti-CD3 bispecific antibodyfor use in a method as disclosed herein before, wherein theanti-PD1/anti-LAG3 bispecific antibody comprises monovalent binding toPD-1 and monovalent binding to LAG3.

In a further aspect, provided is an anti-CD20/anti-CD3 bispecificantibody for use in a method as disclosed herein before, wherein theanti-PD1/anti-LAG3 bispecific antibody is a humanized or chimericantibody. In particular, the anti-PD1/anti-LAG3 bispecific antibody is ahumanized antibody. Furthermore, provided is an anti-PD1/anti-LAG3bispecific antibody as described herein before, wherein theanti-PD1/anti-LAG3 bispecific antibody comprises an Fc domain comprisinga modification promoting the association of the first and second subunitof the Fc domain. In one aspect, provided is an anti-PD1/anti-LAG3bispecific antibody, wherein the first subunit of the Fc domaincomprises knobs and the second subunit of the Fc domain comprises holesaccording to the knobs into holes method. In particular, the firstsubunit of the Fc domain comprises the amino acid substitutions S354Cand T366W (EU numbering) and the second subunit of the Fc domaincomprises the amino acid substitutions Y349C, T366S and Y407V (numberingaccording to Kabat EU index).

In a particular aspect, provided is a anti-CD20/anti-CD3 bispecificantibody for use in a method of treating CD20 expressing cancer, whereinthe anti-PD1/anti-LAG3 bispecific antibody comprises

-   -   (a) a first heavy chain comprising an amino acid sequence of SEQ        ID NO: 35, a first light chain comprising an amino acid sequence        of SEQ ID NO: 36, a second heavy chain comprising an amino acid        sequence of SEQ ID NO: 37, and a second light chain comprising        an amino acid sequence of SEQ ID NO:38, or    -   (b) a first heavy chain comprising an amino acid sequence of SEQ        ID NO: 35, a first light chain comprising an amino acid sequence        of SEQ ID NO: 36, a second heavy chain comprising an amino acid        sequence of SEQ ID NO: 39, and a second light chain comprising        an amino acid sequence of SEQ ID NO:40.

More particularly, the anti-PD1/anti-LAG3 bispecific antibody comprisesa first heavy chain comprising an amino acid sequence of SEQ ID NO: 35,a first light chain comprising an amino acid sequence of SEQ ID NO: 36,a second heavy chain comprising an amino acid sequence of SEQ ID NO: 37,and a second light chain comprising an amino acid sequence of SEQ IDNO:38.

Furthermore, provided is an anti-CD20/anti-CD3 bispecific antibody foruse in a method of treating CD20 expressing cancer, wherein theanti-CD20/anti-CD3 bispecific antibody is for use in combination with ananti-PD1/anti-LAG3 bispecific antibody and wherein theanti-CD20/anti-CD3 bispecific antibody comprises a first antigen bindingdomain comprising a heavy chain variable region (V_(H)CD3) and a lightchain variable region (V_(L)CD3), and a second antigen binding domaincomprising a heavy chain variable region (V_(H)CD20) and a light chainvariable region (V_(L)CD20). In one aspect, the anti-CD20/anti-CD3bispecific antibody comprises first antigen binding domain comprises aheavy chain variable region (VHCD3) comprising CDR-H1 sequence of SEQ IDNO:41, CDR-H2 sequence of SEQ ID NO:42, and CDR-H3 sequence of SEQ IDNO:43; and/or a light chain variable region (V_(L)CD3) comprising CDR-L1sequence of SEQ ID NO:44, CDR-L2 sequence of SEQ ID NO:45, and CDR-L3sequence of SEQ ID NO:46. More particularly, the anti-CD20/anti-CD3bispecific antibody comprises a first antigen binding domain comprises aheavy chain variable region (VHCD3) comprising the amino acid sequenceof SEQ ID NO:47 and/or a light chain variable region (V_(L)CD3)comprising the amino acid sequence of SEQ ID NO:48. In one aspect, theanti-CD20/anti-CD3 bispecific antibody for use in a method of treatingCD20 expressing cancer comprises a second antigen binding domaincomprises a heavy chain variable region (V_(H)CD20) comprising CDR-H1sequence of SEQ ID NO:49, CDR-H2 sequence of SEQ ID NO:50, and CDR-H3sequence of SEQ ID NO:51, and/or a light chain variable region(V_(L)CD20) comprising CDR-L1 sequence of SEQ ID NO:52, CDR-L2 sequenceof SEQ ID NO:53, and CDR-L3 sequence of SEQ ID NO:54. In particular, thesecond antigen binding domain comprises a heavy chain variable region(V_(H)CD20) comprising the amino acid sequence of SEQ ID NO:55 and/or alight chain variable region (V_(L)CD20) comprising the amino acidsequence of SEQ ID NO:56. In a further aspect, the anti-CD20/anti-CD3bispecific antibody for use in a method of treating CD20 expressingcancer comprises a third antigen binding domain that binds to CD20. Inanother aspect, the anti-CD20/anti-CD3 bispecific antibody comprises anFc domain comprising one or more amino acid substitutions that reducebinding to an Fc receptor and/or effector function.

In one particular aspect, the anti-CD20/anti-CD3 bispecific antibody foruse in a method of treating CD20 expressing cancer is glofitamab. Inanother particular aspect, the anti-CD20/anti-CD3 bispecific antibodyfor use in a method of treating CD20 expressing cancer is mosunetuzumab.

In a further aspect, provided is an anti-CD20/anti-CD3 bispecificantibody for use in a method of treating CD20 expressing cancer, whereinthe anti-CD20/anti-CD3 bispecific antibody is used in combination withanti-PD1/anti-LAG3 bispecific antibody and wherein the combination isfor administration at intervals from about one week to three weeks.

In yet another aspect, the anti-CD20/anti-CD3 bispecific antibody is foruse in a method of treating CD20 expressing cancer, wherein apretreatment with an Type II anti-CD20 antibody, preferablyobinutuzumab, is performed prior to the combination treatment, whereinthe period of time between the pretreatment and the combinationtreatment is sufficient for the reduction of B-cells in the individualin response to the Type II anti-CD20 antibody. Preferably, the Type IIanti-CD20 antibody is obinutuzumab.

In one further aspect, provided is a composition comprising ananti-PD1/anti-LAG3 bispecific antibody for use in the treatment of CD20expressing cancer, wherein said treatment comprises administration ofsaid composition comprising an anti-PD1/anti-LAG3 bispecific antibody incombination with a composition comprising an anti-CD20/anti-CD3bispecific antibody, wherein the anti-PD1/anti-LAG3 bispecific antibodycomprises a first antigen binding domain that specifically binds toprogrammed cell death protein 1 (PD1) and a second antigen bindingdomain that specifically binds to Lymphocyte activation gene-3 (LAG3),wherein the first antigen binding domain specifically binding to PD1comprises a VH domain comprising

-   -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1,    -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2,        and    -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID NO:3;        and        a VL domain comprising    -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:4;    -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5,        and    -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

In one aspect, the composition comprises an anti-PD1/anti-LAG3bispecific antibody comprising a first antigen-binding domainspecifically binding to PD1 comprising the VH domain comprising theamino acid sequence of SEQ ID NO: 9 and the VL domain comprising theamino acid sequence of SEQ ID NO: 10. In one further aspect, thecomposition comprises an anti-PD1/anti-LAG3 bispecific antibodycomprises a second antigen binding domain that specifically binds toLAG3 comprising

-   -   (a) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:11,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:            12, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID NO:            13; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:14,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:            15, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO: 16; or    -   (b) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:19,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:20, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:21; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:22,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:23, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:24.

In one aspect, the composition comprises an anti-PD1/anti-LAG3bispecific antibody comprising an antigen-binding domain specificallybinding to LAG3 comprising

-   -   (a) a VH domain comprising the amino acid sequence of SEQ ID NO:        17 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 18, or    -   (b) a VH domain comprising the amino acid sequence of SEQ ID NO:        25 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 26.

In one particular aspect, the composition comprises ananti-PD1/anti-LAG3 bispecific antibody comprising a first Fab fragmentspecifically binding to PD1 comprising a VH domain comprising the aminoacid sequence of SEQ ID NO: 9 and a VL domain comprising the amino acidsequence of SEQ ID NO: 10, and a second Fab fragment specificallybinding to LAG3 comprising a VH domain comprising the amino acidsequence of SEQ ID NO: 17 and a VL domain comprising the amino acidsequence of SEQ ID NO: 18.

Furthermore, provided is a composition comprising an anti-PD1/anti-LAG3bispecific antibody for use in the treatment of CD20 expressing cancer,wherein said treatment comprises administration of said compositioncomprising an anti-PD1/anti-LAG3 bispecific antibody in combination witha composition comprising an anti-CD20/anti-CD3 bispecific antibody,wherein the anti-CD20/anti-CD3 bispecific antibody comprises a firstantigen binding domain comprising a heavy chain variable region(V_(H)CD3) and a light chain variable region (V_(L)CD3), and a secondantigen binding domain comprising a heavy chain variable region(V_(H)CD20) and a light chain variable region (V_(L)CD20). In oneaspect, the anti-CD20/anti-CD3 bispecific antibody comprises firstantigen binding domain comprises a heavy chain variable region (VHCD3)comprising CDR-H1 sequence of SEQ ID NO:41, CDR-H2 sequence of SEQ IDNO:42, and CDR-H3 sequence of SEQ ID NO:43; and/or a light chainvariable region (V_(L)CD3) comprising CDR-L1 sequence of SEQ ID NO:44,CDR-L2 sequence of SEQ ID NO:45, and CDR-L3 sequence of SEQ ID NO:46.More particularly, the anti-CD20/anti-CD3 bispecific antibody comprisesa first antigen binding domain comprises a heavy chain variable region(VHCD3) comprising the amino acid sequence of SEQ ID NO:47 and/or alight chain variable region (V_(L)CD3) comprising the amino acidsequence of SEQ ID NO:48. In one aspect, the anti-CD20/anti-CD3bispecific antibody comprises a second antigen binding domain comprisesa heavy chain variable region (V_(H)CD20) comprising CDR-H1 sequence ofSEQ ID NO:49, CDR-H2 sequence of SEQ ID NO:50, and CDR-H3 sequence ofSEQ ID NO:51, and/or a light chain variable region (V_(L)CD20)comprising CDR-L1 sequence of SEQ ID NO:52, CDR-L2 sequence of SEQ IDNO:53, and CDR-L3 sequence of SEQ ID NO:54. In particular, the secondantigen binding domain comprises a heavy chain variable region(V_(H)CD20) comprising the amino acid sequence of SEQ ID NO:55 and/or alight chain variable region (V_(L)CD20) comprising the amino acidsequence of SEQ ID NO:56. In a further aspect, the anti-CD20/anti-CD3bispecific antibody comprises a third antigen binding domain that bindsto CD20. In another aspect, the anti-CD20/anti-CD3 bispecific antibodycomprises an Fc domain comprising one or more amino acid substitutionsthat reduce binding to an Fc receptor and/or effector function. In oneparticular aspect, the anti-CD20/anti-CD3 bispecific antibody isglofitamab. In another particular aspect, the anti-CD20/anti-CD3bispecific antibody is mosunetuzumab.

In yet another aspect, provided is a composition comprising ananti-PD1/anti-LAG3 bispecific antibody for use in the treatment of CD20expressing cancer, wherein said treatment comprises administration ofsaid composition comprising an anti-PD1/anti-LAG3 bispecific antibody incombination with a composition comprising an anti-CD20/anti-CD3bispecific antibody, wherein a pretreatment with an Type II anti-CD20antibody, preferably obinutuzumab, is performed prior to the combinationtreatment, wherein the period of time between the pretreatment and thecombination treatment is sufficient for the reduction of B-cells in theindividual in response to the Type II anti-CD20 antibody. Preferably,the Type II anti-CD20 antibody is obinutuzumab.

In a further aspect, provided is a pharmaceutical product comprising (A)a first composition comprising as active ingredient ananti-CD20/anti-CD3 bispecific antibody and a pharmaceutically acceptablecarrier; and (B) a second composition comprising as active ingredient ananti-PD1/anti-LAG3 bispecific antibody and a pharmaceutically acceptablecarrier, for use in the combined, sequential or simultaneous, treatmentof a disease, in particular CD20 expressing cancer.

In another aspect, provided is a pharmaceutical composition comprising acombination of an anti-CD20/anti-CD3 bispecific antibody and ananti-PD1/anti-LAG3 bispecific antibody for use in the combined,sequential or simultaneous, treatment of a disease, in particular CD20expressing cancer. In particular, the pharmaceutical composition is foruse in the treatment of B-cell proliferative disorders, in particular adisease selected from the group consisting of Non-Hodgkin lymphoma(NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia(CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL),mantle-cell lymphoma (MCL), marginal zone lymphoma (MZL), Multiplemyeloma (MM) and Hodgkin lymphoma (HL).

In another aspect, provided is the use of a combination of ananti-CD20/anti-CD3 bispecific antibody and an anti-PD1/anti-LAG3bispecific antibody in the manufacture of a medicament for treating ordelaying progression of a proliferative disease, in particular fortreating a CD20 expressing cancer, wherein the anti-PD1/anti-LAG3bispecific antibody comprises a first antigen binding domain thatspecifically binds to programmed cell death protein 1 (PD1) and a secondantigen binding domain that specifically binds to Lymphocyte activationgene-3 (LAG3), wherein the first antigen binding domain specificallybinding to PD1 comprises a VH domain comprising

-   -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1,    -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2,        and    -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID NO:3;        and        a VL domain comprising    -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:4;    -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5,        and    -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

In one further aspect, the anti-PD1/anti-LAG3 bispecific antibodycomprises a second antigen binding domain that specifically binds toLAG3 comprising

-   -   (a) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:11,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:            12, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID NO:            13; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:14,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:            15, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO: 16; or    -   (b) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:19,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:20, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:21; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:22,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:            23, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:24.

In another aspect, provided is the use of a combination of ananti-CD20/anti-CD3 bispecific antibody and an anti-PD1/anti-LAG3bispecific antibody in the manufacture of a medicament for treating ordelaying progression of a proliferative disease, in particular fortreating a CD20 expressing cancer, wherein the anti-PD1/anti-LAG3bispecific antibody comprises a first Fab fragment specifically bindingto PD1 comprising a VH domain comprising the amino acid sequence of SEQID NO: 9 and a VL domain comprising the amino acid sequence of SEQ IDNO: 10, and a second Fab fragment specifically binding to LAG3comprising a VH domain comprising the amino acid sequence of SEQ ID NO:17 and a VL domain comprising the amino acid sequence of SEQ ID NO: 18.

In yet another aspect, provided is the use of a combination of ananti-CD20/anti-CD3 bispecific antibody and an anti-PD1/anti-LAG3bispecific antibody in the manufacture of a medicament for treating ordelaying progression of a proliferative disease, in particular fortreating a CD20 expressing cancer, wherein a pretreatment with an TypeII anti-CD20 antibody, preferably obinutuzumab, is performed prior tothe combination treatment, wherein the period of time between thepretreatment and the combination treatment is sufficient for thereduction of B-cells in the individual in response to the Type IIanti-CD20 antibody. Preferably, the Type II anti-CD20 antibody isobinutuzumab.

In a further aspect, provided is a method for treating CD20 expressingcancer in a subject comprising administering to the subject an effectiveamount of an anti-CD20/anti-CD3 antibody and an effective amount of ananti-PD1/anti-LAG3 bispecific antibody, wherein the anti-PD1/anti-LAG3bispecific antibody comprises a first antigen binding domain thatspecifically binds to programmed cell death protein 1 (PD1) and a secondantigen binding domain that specifically binds to Lymphocyte activationgene-3 (LAG3), wherein the first antigen binding domain specificallybinding to PD1 comprises a VH domain comprising

-   -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1,    -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2,        and    -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID NO:3;        and        a VL domain comprising    -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:4;    -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5,        and    -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

In one aspect, provided is the method, wherein the anti-PD1/anti-LAG3bispecific antibody comprises a second antigen binding domain thatspecifically binds to LAG3 comprising

-   -   (a) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:11,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:            12, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID NO:            13; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:14,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:            15, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO: 16; or    -   (b) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:19,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:20, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:21; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:22,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:23, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:24.

In another aspect, provided is the method, wherein theanti-PD1/anti-LAG3 bispecific antibody comprises a first Fab fragmentspecifically binding to PD1 comprising a VH domain comprising the aminoacid sequence of SEQ ID NO: 9 and a VL domain comprising the amino acidsequence of SEQ ID NO: 10, and a second Fab fragment specificallybinding to LAG3 comprising a VH domain comprising the amino acidsequence of SEQ ID NO: 17 and a VL domain comprising the amino acidsequence of SEQ ID NO: 18.

In one aspect, provided is the method, wherein the anti-CD20/anti-CD3bispecific antibody comprises a first antigen binding domain comprisinga heavy chain variable region (V_(H)CD3) and a light chain variableregion (V_(L)CD3), and a second antigen binding domain comprising aheavy chain variable region (V_(H)CD20) and a light chain variableregion (V_(L)CD20). In one aspect, the anti-CD20/anti-CD3 bispecificantibody comprises first antigen binding domain comprises a heavy chainvariable region (VHCD3) comprising CDR-H1 sequence of SEQ ID NO:41,CDR-H2 sequence of SEQ ID NO:42, and CDR-H3 sequence of SEQ ID NO:43;and/or a light chain variable region (V_(L)CD3) comprising CDR-L1sequence of SEQ ID NO:44, CDR-L2 sequence of SEQ ID NO:45, and CDR-L3sequence of SEQ ID NO:46. More particularly, the anti-CD20/anti-CD3bispecific antibody comprises a first antigen binding domain comprises aheavy chain variable region (VHCD3) comprising the amino acid sequenceof SEQ ID NO:47 and/or a light chain variable region (V_(L)CD3)comprising the amino acid sequence of SEQ ID NO:48. In one aspect, theanti-CD20/anti-CD3 bispecific antibody comprises a second antigenbinding domain comprises a heavy chain variable region (V_(H)CD20)comprising CDR-H1 sequence of SEQ ID NO:49, CDR-H2 sequence of SEQ IDNO:50, and CDR-H3 sequence of SEQ ID NO:51, and/or a light chainvariable region (V_(L)CD20) comprising CDR-L1 sequence of SEQ ID NO:52,CDR-L2 sequence of SEQ ID NO:53, and CDR-L3 sequence of SEQ ID NO:54. Inparticular, the second antigen binding domain comprises a heavy chainvariable region (V_(H)CD20) comprising the amino acid sequence of SEQ IDNO:55 and/or a light chain variable region (V_(L)CD20) comprising theamino acid sequence of SEQ ID NO: 56. In a further aspect, theanti-CD20/anti-CD3 bispecific antibody comprises a third antigen bindingdomain that binds to CD20. In another aspect, the anti-CD20/anti-CD3bispecific antibody comprises an Fc domain comprising one or more aminoacid substitutions that reduce binding to an Fc receptor and/or effectorfunction. In one particular aspect, the anti-CD20/anti-CD3 bispecificantibody is glofitamab. In another particular aspect, theanti-CD20/anti-CD3 bispecific antibody is mosunetuzumab.

In yet another aspect, provided is a method for treating CD20 expressingcancer in a subject comprising administering to the subject an effectiveamount of an anti-CD20/anti-CD3 antibody and an effective amount of ananti-PD1/anti-LAG3 bispecific antibody, wherein a pretreatment with anType II anti-CD20 antibody, preferably obinutuzumab, is performed priorto the combination treatment, wherein the period of time between thepretreatment and the combination treatment is sufficient for thereduction of B-cells in the individual in response to the Type IIanti-CD20 antibody. Preferably, the Type II anti-CD20 antibody isobinutuzumab.

In one aspect, the anti-CD20/anti-CD3 bispecific antibody and theanti-PD1/anti-LAG3 bispecific antibody are administered together in asingle composition or administered separately in two or more differentcompositions. In a further aspect, the anti-CD20/anti-CD3 bispecificantibody and the anti-PD1/anti-LAG3 bispecific antibody are administeredintravenously or subcutaneously. In another aspect, theanti-CD20/anti-CD3 bispecific antibody is administered concurrentlywith, prior to, or subsequently to the anti-PD1/anti-LAG3 bispecificantibody.

In any of the above aspects the subject is preferably a mammal,particularly a human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are schematic illustrations of particularanti-PD1/anti-LAG3 bispecific antibodies (FIG. 1A) and a particularanti-CD20/anti-CD3 bispecific antibody (FIG. 1B) as used in theExamples. These molecules are described in more detail in Examples 2 and1, respectively. FIG. 1A shows the anti-PD1/anti-LAG3 bispecificantibodies in 1+1 format, wherein the PD1 binding domain comprises acrossFab (with VH/VL domain exchange) and the LAG3 binding domaincomprises CHT and CK domains with amino acid mutations to supportcorrect pairing (“charged variants”). The Fc part comprises the knobinto hole mutations (illustrated by the black arrow) and the amino acidmutations L234A, L235A and P329G almost completely abolishing Fcγreceptor binding of the human IgG1 Fc domain. In FIG. 1B an exemplarybispecific anti-CD20/anti-CD3 antibody in 2+1 format is shown (namedCD20 TCB).

FIG. 2 shows the effect of an anti-PD1/anti-LAG3 bispecific antibody(PD1-LAG3 BsAb) in combination with CD20 TCB on cytotoxic Granzyme Brelease by human CD4 T cells cocultured with a B cell-lymphoblatoid cellline (ARH77). PD1-LAG3 BsAb is compared with PD-1 antibodies (nivolumab,pembrolizumab and the parental PD-1 antibody.

FIG. 3 shows the protocol of the in vivo efficacy study of PD1-LAG3 BsAbvs. PD1 antibodies in combination with CD20 TCB in WSU-DLCL2-bearingfully humanized NSG mice. In the table below the subgroups of micereceiving different combinations are defined. The experiment isdescribed in Example 4.

FIG. 4 shows the results of the study. Humanized NSG mice were injecteds.c. with 1.5×10⁶ WSU-DLCL2 cells expressing CD20. After the tumorsreached an average volume of around 350-400 mm³ (on day 14), mice wererandomized to 6 groups receiving: A) phosphate-buffer saline (PBS;vehicle) as control; B) CD20-TCB (0.15 mg/kg once/week i.v.), C)CD20-TCB (0.15 mg/kg once/week i.v.)+Nivolumab (1.5 mg/kg once/weeki.v.), D) CD20-TCB (0.15 mg/kg once/week i.v.)+Nivolumab (1.5 mg/kgonce/week i.p.)+anti-LAG3 (1.5 mg/kg once/week i.v.), E) CD20-TCB (0.15mg/kg once/week i.v.)+PD1-LAG3 BsAb (1.5 mg/kg once/week i.v.), F)CD20-TCB (0.15 mg/kg once/week i.v.)+PD1-LAG3 BsAb (3 mg/kg once/weeki.v.). Tumor volume was measured by digital caliper 3 times a week. Datais shown as average tumor volume and standard error of mean (+/−SEM).

In FIGS. 5A, 5B, 5C, 5D, 5E and 5F the measurements of tumor volumes(mm³+/−SEM), over a period from day 14 to day 45, are shown for eachindividual animal showing homogeneity of anti-tumor response in thegroups treated with PD1-LAG3 BsAb. The tumor growth curves are shown forthe vehicle group in FIG. 5A, for CD20 CD3 TCB alone (0.15 mg/kg) inFIG. 5B, for the combination of CD20 CD3 TCB with Nivolumab (1.5 mg/kg)in FIG. 5C, for the combination of CD20 CD3 TCB with nivolumab (1.5mg/kg) and anti-LAG3 (1.5 mg/kg) in FIG. 5D, for the combination of CD20CD3 TCB with PD1/LAG3 BsAb in FIG. 5E (1.5 mg/kg) and in FIG. 5F (3mg/kg PD1/LAG3 BsAb).

FIG. 6 shows that the combination of CD20 CD3 TCB and PD1/LAG3 BsAb at 3mg/kg resulted in a statistical significant tumor protection as comparedto the treatment in combination with nivolumab or nivolumab+anti-LAG3.For this analysis tumor volume data were transformed introducing a newend point: we evaluated if the last observed tumor volume of each animalwas below 800 mm³ or not providing a binary readout and a percentage oflow size tumor. This endpoint was then subjected to pairwise groupcomparisons based on a Chi² test.

FIG. 7 shows the protocol of the in vivo efficacy study of CD20 TCB incombination with PD1-LAG3 BsAb or with pembrolizumab+anti-LAG3 inOCI-Ly18 bearing fully humanized NSG mice. In the table below thesubgroups of mice receiving different combinations are defined. Theexperiment is described in Example 5.

FIG. 8 shows the results of the study. Humanized NSG mice were injecteds.c. with OCI-Ly18 lymphoma cells expressing CD20. After the tumorsreached an average volume of around 200 mm³ (on day 10), mice wererandomized and the therapies injected. The measurements of tumor volumes(mm³+/−SEM), are shown as mean volume within the group of mice. Thetumor size was measured until there were at least 6 (for vehicle) or 7(for treatment groups) mice/group/timepoint. Vehicle was followed untilday 26 while the treatment groups until day 35. Data is shown as averagetumor volume and standard error of mean (+/−SEM).

In FIGS. 9A, 9B, 9C and 9D the measurements of tumor volumes(mm³+/−SEM), over a period from day 10 to day 35, are shown for eachindividual animal. The tumor growth curves are shown for the vehiclegroup in FIG. 9A, for CD20 CD3 TCB alone in FIG. 9B, for the combinationof CD20 CD3 TCB with PD1-LAG3 BsAb in FIG. 9C, and for the combinationof CD20 CD3 TCB with pembrolizumab and anti-LAG3 in FIG. 9D.

FIG. 10 shows the protocol of the in vivo efficacy study of CD20 TCBalone compared to the combination with PD1-LAG3 BsAb in OCI-Ly18 bearingfully humanized NSG mice, when a pre-treatment with obinutuzumab isused. In the table below the subgroups of mice receiving differentcombinations are defined. The experiment is described in Example 6.

FIG. 11 shows the results of the study. Humanized NSG mice were injecteds.c. with OCI-Ly18 lymphoma cells expressing CD20. After the tumorsreached an average volume of around 400 mm³ (on day 17), mice wererandomized and the therapies injected according to the experimentlayout. The measurements of tumor volumes (mm³+/−SEM), are shown as meanvolume within the group of mice. The tumor size was measured until day35 for treatment groups, while until day 26 for the vehicle group.

In FIGS. 12A, 12B and 12C the measurements of tumor volumes (mm³+/−SEM),over a period from day 17 to day 35, are shown for each individualanimal. The tumor growth curves are shown for the vehicle group in FIG.12A, for obinutuzumab and CD20 CD3 TCB in FIG. 12B, and for thecombination of obinutuzumab and CD20 CD3 TCB with PD1-LAG3 BsAb in FIG.12C.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as generally used in the art to which thisinvention belongs. For purposes of interpreting this specification, thefollowing definitions will apply and whenever appropriate, terms used inthe singular will also include the plural and vice versa.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, monospecific and multispecificantibodies (e.g., bispecific antibodies), and antibody fragments so longas they exhibit the desired antigen-binding activity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g. containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen.

The term “monospecific” antibody as used herein denotes an antibody thathas one or more binding sites each of which bind to the same epitope ofthe same antigen. The term “bispecific” means that the antibody is ableto specifically bind to at least two distinct antigenic determinants,for example two binding sites each formed by a pair of an antibody heavychain variable domain (VH) and an antibody light chain variable domain(VL) binding to different antigens or to different epitopes on the sameantigen. Such a bispecific antibody is a 1+1 format. Other bispecificantibody formats are 2+1 formats (comprising two binding sites for afirst antigen or epitope and one binding site for a second antigen orepitope) or 2+2 formats (comprising two binding sites for a firstantigen or epitope and two binding sites for a second antigen orepitope). Typically, a bispecific antibody comprises two antigen bindingsites, each of which is specific for a different antigenic determinant.

The term “valent” as used within the current application denotes thepresence of a specified number of binding domains in an antigen bindingmolecule. As such, the terms “bivalent”, “tetravalent”, and “hexavalent”denote the presence of two binding domain, four binding domains, and sixbinding domains, respectively, in an antigen binding molecule. Thebispecific antibodies according to the invention are at least “bivalent”and may be “trivalent” or “multivalent” (e.g. “tetravalent” or“hexavalent”). In a particular aspect, the antibodies of the presentinvention have two or more binding sites and are bispecific. That is,the antibodies may be bispecific even in cases where there are more thantwo binding sites (i.e. that the antibody is trivalent or multivalent).

The terms “full length antibody”, “intact antibody”, and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure.“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG-classantibodies are heterotetrameric glycoproteins of about 150,000 daltons,composed of two light chains and two heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3),also called a heavy chain constant region. Similarly, from N- toC-terminus, each light chain has a variable region (VL), also called avariable light domain or a light chain variable domain, followed by alight chain constant domain (CL), also called a light chain constantregion. The heavy chain of an antibody may be assigned to one of fivetypes, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some ofwhich may be further divided into subtypes, e.g. γ1 (IgG1), γ2 (IgG2),γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies, triabodies, tetrabodies, cross-Fab fragments; linearantibodies; single-chain antibody molecules (e.g. scFv); multispecificantibodies formed from antibody fragments and single domain antibodies.For a review of certain antibody fragments, see Hudson et al., Nat Med9, 129-134 (2003). For a review of scFv fragments, see e.g. Pluckthun,in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg andMoore eds., Springer-Verlag, New York, pp. 269-315 (1994); see also WO93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For discussion ofFab and F(ab′)2 fragments comprising salvage receptor binding epitoperesidues and having increased in vivo half-life, see U.S. Pat. No.5,869,046. Diabodies are antibody fragments with two antigen bindingdomains that may be bivalent or bispecific, see, for example, EP404,097; WO 1993/01161; Hudson et al., Nat Med 9, 129-134 (2003); andHollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993).Triabodies and tetrabodies are also described in Hudson et al., Nat Med9, 129-134 (2003). Single-domain antibodies are antibody fragmentscomprising all or a portion of the heavy chain variable domain or all ora portion of the light chain variable domain of an antibody. In certainembodiments, a single-domain antibody is a human single-domain antibody(Domantis, Inc., Waltham, MA; see e.g. U.S. Pat. No. 6,248,516 B1). Inaddition, antibody fragments comprise single chain polypeptides havingthe characteristics of a VH domain, namely being able to assembletogether with a VL domain, or of a VL domain, namely being able toassemble together with a VH domain to a functional antigen binding siteand thereby providing the antigen binding property of full lengthantibodies. Antibody fragments can be made by various techniques,including but not limited to proteolytic digestion of an intact antibodyas well as production by recombinant host cells (e.g. E. coli or phage),as described herein.

Papain digestion of intact antibodies produces two identicalantigen-binding fragments, called “Fab” fragments containing each theheavy- and light-chain variable domains and also the constant domain ofthe light chain and the first constant domain (CH1) of the heavy chain.As used herein, Thus, the term “Fab fragment” refers to an antibodyfragment comprising a light chain fragment comprising a VL domain and aconstant domain of a light chain (CL), and a VH domain and a firstconstant domain (CH1) of a heavy chain. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH1 domain including one or more cysteins from theantibody hinge region. Fab′-SH are Fab′ fragments wherein the cysteineresidue(s) of the constant domains bear a free thiol group. Pepsintreatment yields an F(ab′)₂ fragment that has two antigen-combiningsites (two Fab fragments) and a part of the Fc region.

The term “cross-Fab fragment” or “xFab fragment” or “crossover Fabfragment” refers to a Fab fragment, wherein either the variable regionsor the constant regions of the heavy and light chain are exchanged. Twodifferent chain compositions of a crossover Fab molecule are possibleand comprised in the bispecific antibodies of the invention: On the onehand, the variable regions of the Fab heavy and light chain areexchanged, i.e. the crossover Fab molecule comprises a peptide chaincomposed of the light chain variable region (VL) and the heavy chainconstant region (CH1), and a peptide chain composed of the heavy chainvariable region (VH) and the light chain constant region (CL). Thiscrossover Fab molecule is also referred to as CrossFab_((VLVH)). On theother hand, when the constant regions of the Fab heavy and light chainare exchanged, the crossover Fab molecule comprises a peptide chaincomposed of the heavy chain variable region (VH) and the light chainconstant region (CL), and a peptide chain composed of the light chainvariable region (VL) and the heavy chain constant region (CH1). Thiscrossover Fab molecule is also referred to as CrossFab_((CLCH1)).

A “single chain Fab fragment” or “scFab” is a polypeptide consisting ofan antibody heavy chain variable domain (VH), an antibody constantdomain 1 (CH1), an antibody light chain variable domain (VL), anantibody light chain constant domain (CL) and a linker, wherein saidantibody domains and said linker have one of the following orders inN-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b)VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL;and wherein said linker is a polypeptide of at least 30 amino acids,preferably between 32 and 50 amino acids. Said single chain Fabfragments are stabilized via the natural disulfide bond between the CLdomain and the CH1 domain. In addition, these single chain Fab moleculesmight be further stabilized by generation of interchain disulfide bondsvia insertion of cysteine residues (e.g. position 44 in the variableheavy chain and position 100 in the variable light chain according toKabat numbering).

A “crossover single chain Fab fragment” or “x-scFab” is a is apolypeptide consisting of an antibody heavy chain variable domain (VH),an antibody constant domain 1 (CH1), an antibody light chain variabledomain (VL), an antibody light chain constant domain (CL) and a linker,wherein said antibody domains and said linker have one of the followingorders in N-terminal to C-terminal direction: a) VH-CL-linker-VL-CH1 andb) VL-CH1-linker-VH-CL; wherein VH and VL form together an antigenbinding domain which binds specifically to an antigen and wherein saidlinker is a polypeptide of at least 30 amino acids. In addition, thesex-scFab molecules might be further stabilized by generation ofinterchain disulfide bonds via insertion of cysteine residues (e.g.position 44 in the variable heavy chain and position 100 in the variablelight chain according to Kabat numbering).

A “single-chain variable fragment (scFv)” is a fusion protein of thevariable regions of the heavy (VH) and light chains (V_(L)) of anantibody, connected with a short linker peptide of ten to about 25 aminoacids. The linker is usually rich in glycine for flexibility, as well asserine or threonine for solubility, and can either connect theN-terminus of the V_(H) with the C-terminus of the V_(L), or vice versa.This protein retains the specificity of the original antibody, despiteremoval of the constant regions and the introduction of the linker. scFvantibodies are, e.g. described in Houston, J. S., Methods in Enzymol.203 (1991) 46-96). In addition, antibody fragments comprise single chainpolypeptides having the characteristics of a VH domain, namely beingable to assemble together with a VL domain, or of a VL domain, namelybeing able to assemble together with a VH domain to a functional antigenbinding site and thereby providing the antigen binding property of fulllength antibodies.

“Scaffold antigen binding proteins” are known in the art, for example,fibronectin and designed ankyrin repeat proteins (DARPins) have beenused as alternative scaffolds for antigen-binding domains, see, e.g.,Gebauer and Skerra, Engineered protein scaffolds as next-generationantibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumppet al., Darpins: A new generation of protein therapeutics. DrugDiscovery Today 13: 695-701 (2008). In one aspect of the invention, ascaffold antigen binding protein is selected from the group consistingof CTLA-4 (Evibody), Lipocalins (Anticalin), a Protein A-derivedmolecule such as Z-domain of Protein A (Affibody), an A-domain(Avimer/Maxibody), a serum transferrin (trans-body); a designed ankyrinrepeat protein (DARPin), a variable domain of antibody light chain orheavy chain (single-domain antibody, sdAb), a variable domain ofantibody heavy chain (nanobody, aVH), V_(NAR) fragments, a fibronectin(AdNectin), a C-type lectin domain (Tetranectin); a variable domain of anew antigen receptor beta-lactamase (V_(NAR) fragments), a humangamma-crystallin or ubiquitin (Affilin molecules); a kunitz type domainof human protease inhibitors, microbodies such as the proteins from theknottin family, peptide aptamers and fibronectin (adnectin). CTLA-4(Cytotoxic T Lymphocyte-associated Antigen 4) is a CD28-family receptorexpressed on mainly CD4+ T-cells. Its extracellular domain has avariable domain-like Ig fold. Loops corresponding to CDRs of antibodiescan be substituted with heterologous sequence to confer differentbinding properties. CTLA-4 molecules engineered to have differentbinding specificities are also known as Evibodies (e.g. U.S. Pat. No.7,166,697B1). Evibodies are around the same size as the isolatedvariable region of an antibody (e.g. a domain antibody). For furtherdetails see Journal of Immunological Methods 248 (1-2), 31-45 (2001).Lipocalins are a family of extracellular proteins which transport smallhydrophobic molecules such as steroids, bilins, retinoids and lipids.They have a rigid beta-sheet secondary structure with a number of loopsat the open end of the conical structure which can be engineered to bindto different target antigens. Anticalins are between 160-180 amino acidsin size, and are derived from lipocalins. For further details seeBiochim Biophys Acta 1482: 337-350 (2000), U.S. Pat. No. 7,250,297B1 andUS20070224633. An affibody is a scaffold derived from Protein A ofStaphylococcus aureus which can be engineered to bind to antigen. Thedomain consists of a three-helical bundle of approximately 58 aminoacids. Libraries have been generated by randomization of surfaceresidues. For further details see Protein Eng. Des. Sel. 2004, 17,455-462 and EP 1641818A1. Avimers are multidomain proteins derived fromthe A-domain scaffold family. The native domains of approximately 35amino acids adopt a defined disulfide bonded structure. Diversity isgenerated by shuffling of the natural variation exhibited by the familyof A-domains. For further details see Nature Biotechnology 23(12),1556-1561 (2005) and Expert Opinion on Investigational Drugs 16(6),909-917 (June 2007). A transferrin is a monomeric serum transportglycoprotein. Transferrins can be engineered to bind different targetantigens by insertion of peptide sequences in a permissive surface loop.Examples of engineered transferrin scaffolds include the Trans-body. Forfurther details see J. Biol. Chem 274, 24066-24073 (1999). DesignedAnkyrin Repeat Proteins (DARPins) are derived from Ankyrin which is afamily of proteins that mediate attachment of integral membrane proteinsto the cytoskeleton. A single ankyrin repeat is a 33 residue motifconsisting of two alpha-helices and a beta-turn. They can be engineeredto bind different target antigens by randomizing residues in the firstalpha-helix and a beta-turn of each repeat. Their binding interface canbe increased by increasing the number of modules (a method of affinitymaturation). For further details see J. Mol. Biol. 332, 489-503 (2003),PNAS 100(4), 1700-1705 (2003) and J. Mol. Biol. 369, 1015-1028 (2007)and US20040132028A1.

A single-domain antibody is an antibody fragment consisting of a singlemonomeric variable antibody domain. The first single domains werederived from the variable domain of the antibody heavy chain fromcamelids (nanobodies or V_(H)H fragments). Furthermore, the termsingle-domain antibody includes an autonomous human heavy chain variabledomain (aVH) or V_(NAR) fragments derived from sharks. Fibronectin is ascaffold which can be engineered to bind to antigen. Adnectins consistsof a backbone of the natural amino acid sequence of the 10th domain ofthe 15 repeating units of human fibronectin type III (FN3). Three loopsat one end of the .beta.-sandwich can be engineered to enable anAdnectin to specifically recognize a therapeutic target of interest. Forfurther details see Protein Eng. Des. Sel. 18, 435-444 (2005),US20080139791, WO2005056764 and U.S. Pat. No. 6,818,418B1. Peptideaptamers are combinatorial recognition molecules that consist of aconstant scaffold protein, typically thioredoxin (TrxA) which contains aconstrained variable peptide loop inserted at the active site. Forfurther details see Expert Opin. Biol. Ther. 5, 783-797 (2005).Microbodies are derived from naturally occurring microproteins of 25-50amino acids in length which contain 3-4 cysteine bridges—examples ofmicroproteins include KalataBI and conotoxin and knottins. Themicroproteins have a loop which can beengineered to include upto 25amino acids without affecting the overall fold of the microprotein. Forfurther details of engineered knottin domains, see WO2008098796.

An “antigen binding molecule that binds to the same epitope” as areference molecule refers to an antigen binding molecule that blocksbinding of the reference molecule to its antigen in a competition assayby 50% or more, and conversely, the reference molecule blocks binding ofthe antigen binding molecule to its antigen in a competition assay by50% or more.

As used herein, the term “antigen binding domain” or “antigen-bindingsite” refers to the part of the antigen binding molecule thatspecifically binds to an antigenic determinant. More particularly, theterm “antigen-binding domain” refers the part of an antibody thatcomprises the area which specifically binds to and is complementary topart or all of an antigen. Where an antigen is large, an antigen bindingmolecule may only bind to a particular part of the antigen, which partis termed an epitope. An antigen binding domain may be provided by, forexample, one or more variable domains (also called variable regions).Preferably, an antigen binding domain comprises an antibody light chainvariable region (VL) and an antibody heavy chain variable region (VH).In one aspect, the antigen binding domain is able to bind to its antigenand block or partly block its function. Antigen binding domains thatspecifically bind to PD1 or to LAG3 include antibodies and fragmentsthereof as further defined herein. In addition, antigen binding domainsmay include scaffold antigen binding proteins, e.g. binding domainswhich are based on designed repeat proteins or designed repeat domains(see e.g. WO 2002/020565).

As used herein, the term “antigenic determinant” is synonymous with“antigen” and “epitope,” and refers to a site (e.g. a contiguous stretchof amino acids or a conformational configuration made up of differentregions of non-contiguous amino acids) on a polypeptide macromolecule towhich an antigen binding moiety binds, forming an antigen bindingmoiety-antigen complex. Useful antigenic determinants can be found, forexample, on the surfaces of tumor cells, on the surfaces ofvirus-infected cells, on the surfaces of other diseased cells, on thesurface of immune cells, free in blood serum, and/or in theextracellular matrix (ECM). The proteins useful as antigens herein canbe any native form the proteins from any vertebrate source, includingmammals such as primates (e.g. humans) and rodents (e.g. mice and rats),unless otherwise indicated. In a particular embodiment the antigen is ahuman protein. Where reference is made to a specific protein herein, theterm encompasses the “full-length”, unprocessed protein as well as anyform of the protein that results from processing in the cell. The termalso encompasses naturally occurring variants of the protein, e.g.splice variants or allelic variants.

By “specific binding” is meant that the binding is selective for theantigen and can be discriminated from unwanted or non-specificinteractions. The ability of an antigen binding molecule to bind to aspecific antigen can be measured either through an enzyme-linkedimmunosorbent assay (ELISA) or other techniques familiar to one of skillin the art, e.g. Surface Plasmon Resonance (SPR) technique (analyzed ona BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)),and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).

In one embodiment, the extent of binding of an antigen binding moleculeto an unrelated protein is less than about 10% of the binding of theantigen binding molecule to the antigen as measured, e.g. by SPR. Incertain embodiments, an molecule that binds to the antigen has adissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM,≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁷ M or less, e.g. from 10⁻⁷ M to 10⁻¹³M, e.g. from 10⁻⁹ M to 10⁻¹³ M).

“Affinity” or “binding affinity” refers to the strength of the sum totalof non-covalent interactions between a single binding site of a molecule(e.g. an antibody) and its binding partner (e.g. an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g. antibody and antigen). The affinity of amolecule X for its partner Y can generally be represented by thedissociation constant (Kd), which is the ratio of dissociation andassociation rate constants (koff and kon, respectively). Thus,equivalent affinities may comprise different rate constants, as long asthe ratio of the rate constants remains the same. Affinity can bemeasured by common methods known in the art, including those describedherein. A particular method for measuring affinity is Surface PlasmonResonance (SPR).

As used herein, the term “high affinity” of an antibody refers to anantibody having a Kd of 10⁻⁹ M or less and even more particularly 10⁻¹⁰M or less for a target antigen. The term “low affinity” of an antibodyrefers to an antibody having a Kd of 108 or higher.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

“CD20” refers to B-lymphocyte antigen CD20, also known as B-lymphocytesurface antigen B1 or Leukocyte surface antigen Leu-16, and includes anynative CD20 from any vertebrate source, including mammals such asprimates (e.g. humans) non-human primates (e.g. cynomolgus monkeys) androdents (e.g. mice and rats), unless otherwise indicated. The amino acidsequence of human CD20 is shown in Uniprot accession no. P11836 (version149, SEQ ID NO:61). CD20 is a hydrophobic transmembrane protein with amolecular weight of approximately 35 kD expressed on pre-B and mature Blymphocytes. The corresponding human gene is membrane-spanning4-domains, subfamily A, member 1, also known as MS4A1. This gene encodesa member of the membrane-spanning 4A gene family. Members of thisnascent protein family are characterized by common structural featuresand similar intron/exon splice boundaries and display unique expressionpatterns among hematopoietic cells and nonlymphoid tissues. This geneencodes the B-lymphocyte surface molecule which plays a role in thedevelopment and differentiation of B-cells into plasma cells. Thisfamily member is localized to 11q12, among a cluster of family members.Alternative splicing of this gene results in two transcript variantswhich encode the same protein. The term “CD20” encompasses“full-length,” unprocessed CD20 as well as any form of CD20 that resultsfrom processing in the cell. The term also encompasses naturallyoccurring variants of CD20, e.g., splice variants or allelic variants.

The terms “anti-CD20 antibody” and “an antibody that binds to CD20”refer to an antibody that is capable of binding CD20 with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting CD20. In one embodiment, the extent ofbinding of an anti-CD20 antibody to an unrelated, non-CD20 protein isless than about 10% of the binding of the antibody to CD20 as measured,e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibodythat binds to CD20 has a dissociation constant (Kd) of ≤1 μM, ≤100 nM,≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ M or less,e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). In certainembodiments, an anti-CD20 antibody binds to an epitope of CD20 that isconserved among CD20 from different species.

By “Type II anti-CD20 antibody” is meant an anti-CD20 antibody havingbinding properties and biological activities of Type II anti-CD20antibodies as described in Cragg et al., Blood 103 (2004) 2738-2743;Cragg et al., Blood 101 (2003) 1045-1052, Klein et al., mAbs 5 (2013),22-33, and summarized in Table 1 below.

TABLE A Properties of type I and type II anti-CD20 antibodies type Ianti-CD20 antibodies type II anti-CD20 antibodies Bind class I CD20epitope Bind class II CD20 epitope Localize CD20 to lipid rafts Do notlocalize CD20 to lipid rafts High CDC * Low CDC * ADCC activity * ADCCactivity * Full binding capacity to B cells Approx. half bindingcapacity to B cells Weak homotypic aggregation Homotypic aggregation Lowcell death induction Strong cell death induction * if IgG₁ isotype

Examples of type II anti-CD20 antibodies include e.g. obinutuzumab(GA101), tositumumab (B1), humanized B-Ly1 antibody IgG1 (a chimerichumanized IgG1 antibody as disclosed in WO 2005/044859), 11B8 IgG1 (asdisclosed in WO 2004/035607) and AT80 IgG1.

In one aspect, the Type II anti-CD20 antibody comprises the heavy chainvariable region sequence (VHCD20) of SEQ ID NO: 55 and the light chainvariable region sequence (VLCD20) of SEQ ID NO: 56. In another aspect,the Type II anti-CD20 antibody is engineered to have an increasedproportion of non-fucosylated oligosaccharides in the Fc region ascompared to a non-engineered antibody. In one aspect, at least about 40%of the N-linked oligosaccharides in the Fc region of the Type IIanti-CD20 antibody are non-fucosylated.

In a particular aspect, the Type II anti-CD20 antibody is obinutuzumab(recommended INN, WHO Drug Information, Vol. 26, No. 4, 2012, p. 453).As used herein, obinutuzumab is synonymous for GA101. The tradename isGAZYVA® or GAZYVARO®. This replaces all previous versions (e.g. Vol. 25,No. 1, 2011, p. 75-76), and is formerly known as afutuzumab (recommendedINN, WHO Drug Information, Vol. 23, No. 2, 2009, p. 176; Vol. 22, No. 2,2008, p. 124). In one aspect, the Type II anti-CD20 antibody comprisesthe heavy chain comprising the amino acid sequence of SEQ ID NO:62 andthe light chain comprising the amino acid sequence of SEQ ID NO: 63. Inone aspect, the Type II anti-CD20 antibody is tositumomab.

Examples of type I anti-CD20 antibodies include e.g. rituximab,ofatumumab, veltuzumab, ocaratuzumab, ocrelizumab, PRO131921,ublituximab, HI47 IgG3 (ECACC, hybridoma), 2C6 IgG1 (as disclosed in WO2005/103081), 2F2 IgG1 (as disclosed in WO 2004/035607 and WO2005/103081) and 2H7 IgG1 (as disclosed in WO 2004/056312).

The term “humanized B-Ly1 antibody” refers to humanized B-Ly1 antibodyas disclosed in WO 2005/044859 and WO 2007/031875, which were obtainedfrom the murine monoclonal anti-CD20 antibody B-Ly1 (variable region ofthe murine heavy chain (VH): SEQ ID NO:64; variable region of the murinelight chain (VL): SEQ ID NO:65 (see Poppema, S. and Visser, L., BiotestBulletin 3 (1987) 131-139) by chimerization with a human constant domainfrom IgG1 and following humanization (see WO 2005/044859 and WO2007/031875). These “humanized B-Ly1 antibodies” are disclosed in detailin WO 2005/044859 and WO 2007/031875.

The term “reduction” (and grammatical variations thereof such as“reduce” or “reducing”), for example reduction of the number of B cellsor cytokine release, refers to a decrease in the respective quantity, asmeasured by appropriate methods known in the art. For clarity the termincludes also reduction to zero (or below the detection limit of theanalytical method), i.e. complete abolishment or elimination.Conversely, “increased” refers to an increase in the respectivequantity.

A “T-cell antigen” as used herein refers to an antigenic determinantpresented on the surface of a T lymphocyte, particularly a cytotoxic Tlymphocyte.

A “T cell activating therapeutic agent” as used herein refers to atherapeutic agent capable of inducing T cell activation in a subject,particularly a therapeutic agent designed for inducing T-cell activationin a subject. Examples of T cell activating therapeutic agents includebispecific antibodies that specifically bind an activating T cellantigen, such as CD3, and a target cell antigen, such as CD20 or CD19.Further examples include chimeric antigen receptors (CARs) whichcomprise a T cell activating domain and an antigen binding moiety thatspecifically binds to a target cell antigen, such as CD20 or CD19.

An “activating T cell antigen” as used herein refers to an antigenicdeterminant expressed by a T lymphocyte, particularly a cytotoxic Tlymphocyte, which is capable of inducing or enhancing T cell activationupon interaction with an antigen binding molecule. Specifically,interaction of an antigen binding molecule with an activating T cellantigen may induce T cell activation by triggering the signaling cascadeof the T cell receptor complex. An exemplary activating T cell antigenis CD3.

The term “CD3” refers to any native CD3 from any vertebrate source,including mammals such as primates (e.g. humans), non-human primates(e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unlessotherwise indicated. The term encompasses “full-length,” unprocessed CD3as well as any form of CD3 that results from processing in the cell. Theterm also encompasses naturally occurring variants of CD3, e.g., splicevariants or allelic variants. In one embodiment, CD3 is human CD3,particularly the epsilon subunit of human CD3 (CD3c). The amino acidsequence of human CD3ε is shown in UniProt (www.uniprot.org) accessionno. P07766 (version 144), or NCBI (www.ncbi.nlm.nih.gov/) RefSeqNP_000724.1. See also SEQ ID NO: 66. The amino acid sequence ofcynomolgus [Macaca fascicularis] CD3ε is shown in NCBI GenBank no.BAB71849.1. See also SEQ ID NO: 67.

The term “a bispecific antibody comprising a first antigen bindingdomain that specifically binds to PD1 and a second antigen bindingdomain that specifically binds to LAG3” “a bispecific antibody thatspecifically binds PD1 and LAG3”, “bispecific antigen binding moleculespecific for PD1 and LAG3” or an “anti-PD1/anti-LAG3 antibody” are usedinterchangeably herein and refer to a bispecific antibody that iscapable of binding PD1 and LAG3 with sufficient affinity such that theantibody is useful as a diagnostic and/or therapeutic agent in targetingPD1 and LAG3.

The term “PD1”, also known as Programmed cell death protein 1, is a typeI membrane protein of 288 amino acids that was first described in 1992(Ishida et al., EMBO J., 11 (1992), 3887-3895). PD-1 is a member of theextended CD28/CTLA-4 family of T cell regulators and has two ligands,PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273). The protein's structureincludes an extracellular IgV domain followed by a transmembrane regionand an intracellular tail. The intracellular tail contains twophosphorylation sites located in an immunoreceptor tyrosine-basedinhibitory motif and an immunoreceptor tyrosine-based switch motif,which suggests that PD-1 negatively regulates TCR signals. This isconsistent with binding of SHP-1 and SHP-2 phosphatases to thecytoplasmic tail of PD-1 upon ligand binding. While PD-1 is notexpressed on naïve T cells, it is upregulated following T cell receptor(TCR)-mediated activation and is observed on both activated andexhausted T cells (Agata et al., Int. Immunology 8 (1996), 765-772).These exhausted T-cells have a dysfunctional phenotype and are unable torespond appropriately. Although PD-1 has a relatively wide expressionpattern its most important role is likely as a coinhibitory receptor onT cells (Chinai et al, Trends in Pharmacological Sciences 36 (2015),587-595). Current therapeutic approaches thus focus on blocking theinteraction of PD-1 with its ligands to enhance T cell response. Theterms “Programmed Death 1,” “Programmed Cell Death 1,” “Protein PD-1,”“PD-1,” PD1,” “PDCD1,” “hPD-1” and “hPD-I” can be used interchangeably,and include variants, isoforms, species homologs of human PD-1, andanalogs having at least one common epitope with PD-1. The amino acidsequence of human PD1 is shown in UniProt (www.uniprot.org) accessionno. Q15116 (SEQ ID NO:68).

The terms “anti-PD1 antibody” and “an antibody comprising an antigenbinding domain that binds to PD1” refer to an antibody that is capableof binding PD1, especially a PD1 polypeptide expressed on a cellsurface, with sufficient affinity such that the antibody is useful as adiagnostic and/or therapeutic agent in targeting PD1. In one aspect, theextent of binding of an anti-PD1 antibody to an unrelated, non-PD1protein is less than about 10% of the binding of the antibody to PD1 asmeasured, e.g., by radioimmunoassay (RIA) or flow cytometry (FACS) or bya Surface Plasmon Resonance assay using a biosensor system such as aBiacore® system. In certain aspects, an antigen binding protein thatbinds to human PD1 has a K_(D) value of the binding affinity for bindingto human PD1 of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or≤0.001 nM (e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from10⁻⁹ M to 10⁻¹³ M). In one preferred embodiment the respective K_(D)value of the binding affinities is determined in a Surface PlasmonResonance assay using the Extracellular domain (ECD) of human PD1(PD1-ECD) for the PD1 binding affinity. The term “anti-PD1 antibody”also encompasses bispecific antibodies that are capable of binding PD1and a second antigen.

In a specific aspect, the anti-PD1 antibody is selected from the groupconsisting of MDX 1106 (nivolumab), MK-3475 (pembrolizumab), CT-011(pidilizumab), PDR001 (spartalizumab), SHR1210 (camrelizumab), MEDI-0680(AMP-514), REGN2810, and BGB-108. In one particular aspect, the anti-PD1antibody is pembrolizumab, or an antibody comprising a heavy chaincomprising the amino acid sequence of SEQ ID NO:75 and a light chaincomprising the amino acid sequence of SEQ ID NO:76. Pembrolizumab(Merck), also known as MK-3475, Merck 3475, lambrolizumab, SCH-900475,and KEYTRUDA®, is an anti-PD-1 antibody described in WO 2009/114335 (CASReg. No. 1374853-91-4). In one particular aspect, the anti-PD1 antibodyis nivolumab, or an antibody comprising a heavy chain comprising theamino acid sequence of SEQ ID NO:77 and a light chain comprising theamino acid sequence of SEQ ID NO:78. Nivolumab (CAS Registry Number:946414-94-4, Bristol-Myers Squibb/Ono), also known as MDX-1106-04,MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibodydescribed in WO 2006/121168 (CAS Reg. No. 946414-94-4). In anotherparticular aspect, the anti-PD-1 antibody comprising a heavy chainvariable domain VH comprising the amino acid sequence of SEQ ID NO:7 anda light chain variable domain VL comprising the amino acid sequence ofSEQ ID NO:8, or a humanized variant thereof. In a particular aspect, theanti-PD-1 antibody comprising a heavy chain variable domain VHcomprising the amino acid sequence of SEQ ID NO:9 and a light chainvariable domain VL comprising the amino acid sequence of SEQ ID NO:10.

The terms “LAG3” or “Lag-3” or “Lymphocyte activation gene-3” or “CD223”as used herein refer to any native LAG3 from any vertebrate source,including mammals such as primates (e.g. humans) and rodents (e.g., miceand rats), unless otherwise indicated. The term encompasses“full-length,” unprocessed LAG3 as well as any form of LAG3 resultingfrom processing in the cell. The term also encompasses naturallyoccurring variants of LAG3, e.g., splice variants or allelic variants.In one preferred embodiment the term “LAG3” refers to human LAG3. Theamino acid sequence of an exemplary processed (without signal sequences)LAG3 is shown in SEQ ID NO:69. The amino acid sequence of an exemplaryExtracellular Domain (ECD) LAG3 is shown in SEQ ID NO:70.

The terms “anti-LAG3 antibody” and “an antibody that binds to LAG3”refer to an antibody that is capable of binding LAG3 with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting LAG3. In one aspect, the extent ofbinding of an anti-LAG3 antibody to an unrelated, non-LAG3 protein isless than about 10% of the binding of the antibody to LAG3 as measured,e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibodythat binds to LAG3 has a dissociation constant (Kd) of ≤1 μM, ≤100 nM,≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ M or less,e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). In certainaspects, an anti-LAG3 antibody binds to an epitope of LAG3 that isconserved among LAG3 from different species. In one preferredembodiment, an “anti-LAG3 antibody”, “an antibody that specificallybinds to human LAG3”, and “an antibody that binds to human LAG3” refersto an antibody specifically binding to the human LAG3 antigen or itsExtracellular Domain (ECD) with a binding affinity of a K_(D)-value of1.0×10⁻⁸ mol/l or lower, in one embodiment of a K_(D)-value of 1.0×10⁻⁹mol/l or lower, in one embodiment of a K_(D)-value of 1.0×10⁻⁹ mol/l to1.0×10⁻¹³ mol/l. In this context the binding affinity is determined witha standard binding assay, such as surface plasmon resonance technique(BIAcore®, GE-Healthcare Uppsala, Sweden) e.g. using the LAG3extracellular domain. The term “anti-LAG3 antibody” also encompassesbispecific antibodies that are capable of binding LAG3 and a secondantigen. In one aspect, the anti-LAG3 antibody is relatlimab orBMS-986016, or an antibody comprising a heavy chain variable domaincomprising the amino acid sequence of SEQ ID NO:27 and a light chainvariable domain comprising the amino acid sequence of SEQ ID NO:28.

A “blocking” antibody or an “antagonist” antibody is one that inhibitsor reduces a biological activity of the antigen it binds. In someembodiments, blocking antibodies or antagonist antibodies substantiallyor completely inhibit the biological activity of the antigen. Forexample, the bispecific antibodies of the invention block the signalingthrough PD-1 and LAG3 so as to restore a functional response by T cells(e.g., proliferation, cytokine production, target cell killing) from adysfunctional state to antigen stimulation.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding the antigenbinding molecule to antigen. The variable domains of the heavy chain andlight chain (VH and VL, respectively) of a native antibody generallyhave similar structures, with each domain comprising four conservedframework regions (FRs) and three hypervariable regions (HVRs). See,e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page91 (2007). A single VH or VL domain may be sufficient to conferantigen-binding specificity.

The term “hypervariable region” or “HVR” as used herein refers to eachof the regions of an antibody variable domain which are hypervariable insequence and which determine antigen binding specificity, for example“complementarity determining regions” (“CDRs”). Generally, antibodiescomprise six CDRs: three in the VH (CDR-H1, CDR-H2, CDR-H3), and threein the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include:

-   -   (a) hypervariable loops occurring at amino acid residues 26-32        (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101        (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));    -   (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56        (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3)        (Kabat et al., Sequences of Proteins of Immunological Interest,        5th Ed. Public Health Service, National Institutes of Health,        Bethesda, MD (1991)); and    -   (c) antigen contacts occurring at amino acid residues 27c-36        (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and        93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745        (1996)).

Unless otherwise indicated, the CDRs are determined according to Kabatet al., supra. One of skill in the art will understand that the CDRdesignations can also be determined according to Chothia, supra,McCallum, supra, or any other scientifically accepted nomenclaturesystem.

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat,” and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al. Usingthis numbering system, the actual linear amino acid sequence may containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or HVR of the variable domain. For example, a heavychain variable domain may include a single amino acid insert (residue52a according to Kabat) after residue 52 of H2 and inserted residues(e.g., residues 82a, 82b, and 82c, etc., according to Kabat) after heavychain FR residue 82. The Kabat numbering of residues may be determinedfor a given antibody by alignment at regions of homology of the sequenceof the antibody with a “standard” Kabat numbered sequence. Generally,native four-chain antibodies comprise six HVRs; three in the VH (H1, H2,H3), and three in the VL (L1, L2, L3).

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g. IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ respectively.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization. Other forms of “humanized antibodies” encompassed by thepresent invention are those in which the constant region has beenadditionally modified or changed from that of the original antibody togenerate the properties according to the invention, especially in regardto C1q binding and/or Fc receptor (FcR) binding.

A “human” antibody is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

The term “Fc domain” or “Fe region” herein is used to define aC-terminal region of an antibody heavy chain that contains at least aportion of the constant region. The term includes native sequence Fcregions and variant Fc regions. Particularly, a human IgG heavy chain Fcregion extends from Cys226, or from Pro230, to the carboxyl-terminus ofthe heavy chain. However, the C-terminal lysine (Lys447) of the Fcregion may or may not be present. The amino acid sequences of the heavychains are always presented with the C-terminal lysine, however variantswithout the C-terminal lysine are included in the invention.

An IgG Fc region comprises an IgG CH2 and an IgG CH3 domain. The “CH2domain” of a human IgG Fc region usually extends from an amino acidresidue at about position 231 to an amino acid residue at about position340. In one embodiment, a carbohydrate chain is attached to the CH2domain. The CH2 domain herein may be a native sequence CH2 domain orvariant CH2 domain. The “CH3 domain” comprises the stretch of residuesC-terminal to a CH2 domain in an Fc region (i.e. from an amino acidresidue at about position 341 to an amino acid residue at about position447 of an IgG). The CH3 region herein may be a native sequence CH3domain or a variant CH3 domain (e.g. a CH3 domain with an introduced“protuberance” (“knob”) in one chain thereof and a correspondingintroduced “cavity” (“hole”) in the other chain thereof; see U.S. Pat.No. 5,821,333, expressly incorporated herein by reference). Such variantCH3 domains may be used to promote heterodimerization of twonon-identical antibody heavy chains as herein described. Unlessotherwise specified herein, numbering of amino acid residues in the Fcregion or constant region is according to the EU numbering system, alsocalled the EU index, as described in Kabat et al., Sequences of Proteinsof Immunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, M D, 1991.

The “knob-into-hole” technology is described e.g. in U.S. Pat. Nos.5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) andCarter, J Immunol Meth 248, 7-15 (2001). Generally, the method involvesintroducing a protuberance (“knob”) at the interface of a firstpolypeptide and a corresponding cavity (“hole”) in the interface of asecond polypeptide, such that the protuberance can be positioned in thecavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory cavities of identicalor similar size to the protuberances are created in the interface of thesecond polypeptide by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine). The protuberance and cavitycan be made by altering the nucleic acid encoding the polypeptides, e.g.by site-specific mutagenesis, or by peptide synthesis. In a specificembodiment a knob modification comprises the amino acid substitutionT366W in one of the two subunits of the Fc domain, and the holemodification comprises the amino acid substitutions T366S, L368A andY407V in the other one of the two subunits of the Fc domain. In afurther specific embodiment, the subunit of the Fc domain comprising theknob modification additionally comprises the amino acid substitutionS354C, and the subunit of the Fc domain comprising the hole modificationadditionally comprises the amino acid substitution Y349C. Introductionof these two cysteine residues results in the formation of a disulfidebridge between the two subunits of the Fc region, thus furtherstabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

A “region equivalent to the Fc region of an immunoglobulin” is intendedto include naturally occurring allelic variants of the Fc region of animmunoglobulin as well as variants having alterations which producesubstitutions, additions, or deletions but which do not decreasesubstantially the ability of the immunoglobulin to mediate effectorfunctions (such as antibody-dependent cellular cytotoxicity). Forexample, one or more amino acids can be deleted from the N-terminus orC-terminus of the Fc region of an immunoglobulin without substantialloss of biological function. Such variants can be selected according togeneral rules known in the art so as to have minimal effect on activity(see, e.g., Bowie, J. U. et al., Science 247:1306-10 (1990)).

The term “effector functions” refers to those biological activitiesattributable to the Fc region of an antibody, which vary with theantibody isotype. Examples of antibody effector functions include: C1qbinding and complement dependent cytotoxicity (CDC), Fc receptorbinding, antibody-dependent cell-mediated cytotoxicity (ADCC),antibody-dependent cellular phagocytosis (ADCP), cytokine secretion,immune complex-mediated antigen uptake by antigen presenting cells, downregulation of cell surface receptors (e.g. B cell receptor), and B cellactivation.

An “activating Fc receptor” is an Fc receptor that following engagementby an Fc region of an antibody elicits signaling events that stimulatethe receptor-bearing cell to perform effector functions. Activating Fcreceptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), andFcαRI (CD89). A particular activating Fc receptor is human FcγRIIIa (seeUniProt accession no. P08637, version 141).

The term “peptide linker” refers to a peptide comprising one or moreamino acids, typically about 2 to 20 amino acids. Peptide linkers areknown in the art or are described herein. Suitable, non-immunogeniclinker peptides are, for example, (G₄S)₁, (SG₄)₁ or G₄(SG₄)₁ peptidelinkers, wherein “n” is generally a number between 1 and 10, typicallybetween 2 and 4, in particular 2. Peptide linkers of particular interestare (G4S) (SEQ ID NO:71), (G₄S)₂ or GGGGSGGGGS (SEQ ID NO:72), (G4S)₃(SEQ ID NO:73) and (G₄S)₄ (SEQ ID NO:74), more particularly (G₄S)₂ orGGGGSGGGGS (SEQ ID NO:72).

By “fused to” or “connected to” is meant that the components (e.g. anantigen binding domain and a FC domain) are linked by peptide bonds,either directly or via one or more peptide linkers.

The term “amino acid” as used within this application denotes the groupof naturally occurring carboxy α-amino acids comprising alanine (threeletter code: ala, one letter code: A), arginine (arg, R), asparagine(asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q),glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine(ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M),phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine(thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide (protein) sequence is defined as the percentage of aminoacid residues in a candidate sequence that are identical with the aminoacid residues in the reference polypeptide sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN. SAWIor Megalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for aligning sequences, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. For purposes herein, however, % amino acidsequence identity values are generated using the sequence comparisoncomputer program ALIGN-2. The ALIGN-2 sequence comparison computerprogram was authored by Genentech, Inc., and the source code has beenfiled with user documentation in the U.S. Copyright Office, WashingtonD.C., 20559, where it is registered under U.S. Copyright RegistrationNo. TXU510087. The ALIGN-2 program is publicly available from Genentech,Inc., South San Francisco, California, or may be compiled from thesource code. The ALIGN-2 program should be compiled for use on a UNIXoperating system, including digital UNIX V4.0D. All sequence comparisonparameters are set by the ALIGN-2 program and do not vary. In situationswhere ALIGN-2 is employed for amino acid sequence comparisons, the %amino acid sequence identity of a given amino acid sequence A to, with,or against a given amino acid sequence B (which can alternatively bephrased as a given amino acid sequence A that has or comprises a certain% amino acid sequence identity to, with, or against a given amino acidsequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

In certain aspects, amino acid sequence variants of the bispecificantibodies of the invention provided herein are contemplated. Forexample, it may be desirable to improve the binding affinity and/orother biological properties of the bispecific antibodies. Amino acidsequence variants of the bispecific antibodies may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the molecules, or by peptide synthesis.

Such modifications include, for example, deletions from, and/orinsertions into and/or substitutions of residues within the amino acidsequences of the antibody. Any combination of deletion, insertion, andsubstitution can be made to arrive at the final construct, provided thatthe final construct possesses the desired characteristics, e.g.,antigen-binding. Sites of interest for substitutional mutagenesisinclude the HVRs and Framework (FRs). Conservative substitutions areprovided in Table C under the heading “Preferred Substitutions” andfurther described below in reference to amino acid side chain classes(1) to (6). Amino acid substitutions may be introduced into the moleculeof interest and the products screened for a desired activity, e.g.,retained/improved antigen binding, decreased immunogenicity, or improvedADCC or CDC.

TABLE B Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

The term “amino acid sequence variants” includes substantial variantswherein there are amino acid substitutions in one or more hypervariableregion residues of a parent antigen binding molecule (e.g. a humanizedor human antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antigen binding molecule and/or will havesubstantially retained certain biological properties of the parentantigen binding molecule. An exemplary substitutional variant is anaffinity matured antibody, which may be conveniently generated, e.g.,using phage display-based affinity maturation techniques such as thosedescribed herein. Briefly, one or more HVR residues are mutated and thevariant antigen binding molecules displayed on phage and screened for aparticular biological activity (e.g. binding affinity). In certainembodiments, substitutions, insertions, or deletions may occur withinone or more HVRs so long as such alterations do not substantially reducethe ability of the antigen binding molecule to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. A useful method for identification of residues orregions of an antibody that may be targeted for mutagenesis is called“alanine scanning mutagenesis” as described by Cunningham and Wells(1989) Science, 244:1081-1085. In this method, a residue or group oftarget residues (e.g., charged residues such as Arg, Asp, His, Lys, andGlu) are identified and replaced by a neutral or negatively chargedamino acid (e.g., alanine or polyalanine) to determine whether theinteraction of the antibody with antigen is affected. Furthersubstitutions may be introduced at the amino acid locationsdemonstrating functional sensitivity to the initial substitutions.Alternatively, or additionally, a crystal structure of anantigen-antigen binding molecule complex to identify contact pointsbetween the antibody and antigen. Such contact residues and neighboringresidues may be targeted or eliminated as candidates for substitution.Variants may be screened to determine whether they contain the desiredproperties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includebispecific antibodies with an N-terminal methionyl residue. Otherinsertional variants of the molecule include the fusion to the N- orC-terminus to a polypeptide which increases the serum half-life of thebispecific antibody.

In certain aspects, the bispecific antibodies provided herein arealtered to increase or decrease the extent to which the antibody isglycosylated. Glycosylation variants of the molecules may beconveniently obtained by altering the amino acid sequence such that oneor more glycosylation sites is created or removed, e.g. thecarbohydrates attached to the Fc domain may be altered. Nativeantibodies produced by mammalian cells typically comprise a branched,biantennary oligosaccharide that is generally attached by an N-linkageto Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al.TIBTECH 15:26-32 (1997). The oligosaccharide may include variouscarbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose,and sialic acid, as well as a fucose attached to a GlcNAc in the “stem”of the biantennary oligosaccharide structure. In some embodiments,modifications of the oligosaccharide in the bispecific antibodies of theinvention may be made in order to create variants with certain improvedproperties. In one aspect, variants of bispecific antibodies areprovided having a carbohydrate structure that lacks fucose attached(directly or indirectly) to an Fc region. Such fucosylation variants mayhave improved ADCC function, see e.g. US Patent Publication Nos. US2003/0157108 (Presta, L.) or US 2004/0093621 (Kyowa Hakko Kogyo Co.,Ltd). Further variants of the bispecific antibodies of the inventioninclude those with bisected oligosaccharides, e.g., in which abiantennary oligosaccharide attached to the Fc region is bisected byGlcNAc. Such variants may have reduced fucosylation and/or improved ADCCfunction, see for example WO 2003/011878 (Jean-Mairet et al.); U.S. Pat.No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.).Variants with at least one galactose residue in the oligosaccharideattached to the Fc region are also provided. Such antibody variants mayhave improved CDC function and are described, e.g., in WO 1997/30087(Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

In certain aspects, it may be desirable to create cysteine engineeredvariants of the bispecific antibodies of the invention, e.g.,“thioMAbs,” in which one or more residues of the molecule aresubstituted with cysteine residues. In particular embodiments, thesubstituted residues occur at accessible sites of the molecule. Bysubstituting those residues with cysteine, reactive thiol groups arethereby positioned at accessible sites of the antibody and may be usedto conjugate the antibody to other moieties, such as drug moieties orlinker-drug moieties, to create an immunoconjugate. In certainembodiments, any one or more of the following residues may besubstituted with cysteine: V205 (Kabat numbering) of the light chain;A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of theheavy chain Fc region. Cysteine engineered antigen binding molecules maybe generated as described, e.g., in U.S. Pat. No. 7,521,541.

In certain aspects, the bispecific antibodies provided herein may befurther modified to contain additional non-proteinaceous moieties thatare known in the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer isattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether thebispecific antibody derivative will be used in a therapy under definedconditions, etc.

In another aspect, conjugates of an antibody and non-proteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the non-proteinaceous moiety is a carbonnanotube (Kam, N. W. et al., Proc. Natl. Acad. Sci. USA 102 (2005)11600-11605). The radiation may be of any wavelength, and includes, butis not limited to, wavelengths that do not harm ordinary cells, butwhich heat the non-proteinaceous moiety to a temperature at which cellsproximal to the antibody-non-proteinaceous moiety are killed.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

The term “polynucleotide” refers to an isolated nucleic acid molecule orconstruct, e.g. messenger RNA (mRNA), virally-derived RNA, or plasmidDNA (pDNA). A polynucleotide may comprise a conventional phosphodiesterbond or a non-conventional bond (e.g. an amide bond, such as found inpeptide nucleic acids (PNA). The term “nucleic acid molecule” refers toany one or more nucleic acid segments, e.g. DNA or RNA fragments,present in a polynucleotide.

By “isolated” nucleic acid molecule or polynucleotide is intended anucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. For example, a recombinant polynucleotide encoding apolypeptide contained in a vector is considered isolated for thepurposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. An isolated polynucleotide includes apolynucleotide molecule contained in cells that ordinarily contain thepolynucleotide molecule, but the polynucleotide molecule is presentextrachromosomally or at a chromosomal location that is different fromits natural chromosomal location. Isolated RNA molecules include in vivoor in vitro RNA transcripts of the present invention, as well aspositive and negative strand forms, and double-stranded forms. Isolatedpolynucleotides or nucleic acids according to the present inventionfurther include such molecules produced synthetically. In addition, apolynucleotide or a nucleic acid may be or may include a regulatoryelement such as a promoter, ribosome binding site, or a transcriptionterminator.

By a nucleic acid or polynucleotide having a nucleotide sequence atleast, for example, 95% “identical” to a reference nucleotide sequenceof the present invention, it is intended that the nucleotide sequence ofthe polynucleotide is identical to the reference sequence except thatthe polynucleotide sequence may include up to five point mutations pereach 100 nucleotides of the reference nucleotide sequence. In otherwords, to obtain a polynucleotide having a nucleotide sequence at least95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. These alterations of the reference sequence may occur at the5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence. As a practical matter,whether any particular polynucleotide sequence is at least 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of thepresent invention can be determined conventionally using known computerprograms, such as the ones discussed above for polypeptides (e.g.ALIGN-2).

The term “expression cassette” refers to a polynucleotide generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in atarget cell. The recombinant expression cassette can be incorporatedinto a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, ornucleic acid fragment. Typically, the recombinant expression cassetteportion of an expression vector includes, among other sequences, anucleic acid sequence to be transcribed and a promoter. In certainembodiments, the expression cassette of the invention comprisespolynucleotide sequences that encode bispecific antigen bindingmolecules of the invention or fragments thereof.

The term “vector” or “expression vector” is synonymous with “expressionconstruct” and refers to a DNA molecule that is used to introduce anddirect the expression of a specific gene to which it is operablyassociated in a target cell. The term includes the vector as aself-replicating nucleic acid structure as well as the vectorincorporated into the genome of a host cell into which it has beenintroduced. The expression vector of the present invention comprises anexpression cassette. Expression vectors allow transcription of largeamounts of stable mRNA. Once the expression vector is inside the targetcell, the ribonucleic acid molecule or protein that is encoded by thegene is produced by the cellular transcription and/or translationmachinery. In one embodiment, the expression vector of the inventioncomprises an expression cassette that comprises polynucleotide sequencesthat encode bispecific antigen binding molecules of the invention orfragments thereof.

The terms “host cell”, “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.A host cell is any type of cellular system that can be used to generatethe bispecific antigen binding molecules of the present invention. Inparticular, the host cell is a prokaryotic or eukaryotic host cell. Hostcells include cultured cells, e.g. mammalian cultured cells, such as CHOcells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mousemyeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells,insect cells, and plant cells, to name only a few, but also cellscomprised within a transgenic animal, transgenic plant or cultured plantor animal tissue.

An “effective amount” of an agent refers to the amount that is necessaryto result in a physiological change in the cell or tissue to which it isadministered.

A “therapeutically effective amount” of an agent, e.g. a pharmaceuticalcomposition, refers to an amount effective, at dosages and for periodsof time necessary, to achieve the desired therapeutic or prophylacticresult. A therapeutically effective amount of an agent for exampleeliminates, decreases, delays, minimizes or prevents adverse effects ofa disease.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g. cows, sheep, cats, dogs, andhorses), primates (e.g. humans and non-human primates such as monkeys),rabbits, and rodents (e.g. mice and rats). Particularly, the individualor subject is a human.

The term “pharmaceutical composition” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable excipient” refers to an ingredient in apharmaceutical composition, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable excipient includes,but is not limited to, a buffer, a stabilizer, or a preservative.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, the moleculesof the invention are used to delay development of a disease or to slowthe progression of a disease.

The term “cancer” as used herein includes lymphomas, lymphocyticleukemias, lung cancer, non small cell lung (NSCL) cancer,bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer,skin cancer, cancer of the head or neck, cutaneous or intraocularmelanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of theanal region, stomach cancer, gastric cancer, colon cancer, breastcancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma ofthe endometrium, carcinoma of the cervix, carcinoma of the vagina,carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus,cancer of the small intestine, cancer of the endocrine system, cancer ofthe thyroid gland, cancer of the parathyroid gland, cancer of theadrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer ofthe penis, prostate cancer, cancer of the bladder, cancer of the kidneyor ureter, renal cell carcinoma, carcinoma of the renal pelvis,mesothelioma, hepatocellular cancer, biliary cancer, neoplasms of thecentral nervous system (CNS), spinal axis tumors, brain stem glioma,glioblastoma multiforme, astrocytomas, schwanomas, ependymomas,medulloblastomas, meningiomas, squamous cell carcinomas, pituitaryadenoma, including refractory versions of any of the above cancers, or acombination of one or more of the above cancers. In one embodiment, theterm cancer refers to a CD20 expressing cancer.

The term “expression of CD20” is intended to indicate an significantlevel of expression CD20 in a cell, preferably on the cell surface of aT- or B-cell, more preferably a B-cell, from a tumor or cancer,respectively, preferably a non-solid tumor. Patients having a “CD20expressing cancer” can be determined by standard assays known in theart. For example CD20 antigen expression can be measured usingimmunohistochemical (IHC) detection, FACS or via PCR-based detection ofthe corresponding mRNA.

The term “CD20 expressing cancer” as used herein refers to all cancersin which the cancer cells show an expression of the CD20 antigen.Preferably CD20 expressing cancer as used herein refers to lymphomas(preferably B-Cell Non-Hodgkin's lymphomas (NHL)) and lymphocyticleukemias. Such lymphomas and lymphocytic leukemias include e.g. a)follicular lymphomas, b) Small Non-Cleaved Cell Lymphomas/Burkitt'slymphoma (including endemic Burkitt's lymphoma, sporadic Burkitt'slymphoma and Non-Burkitt's lymphoma), c) marginal zone lymphomas(including extranodal marginal zone B cell lymphoma (Mucosa-associatedlymphatic tissue lymphomas, MALT), nodal marginal zone B cell lymphomaand splenic marginal zone lymphoma), d) Mantle cell lymphoma (MCL), e)Large Cell Lymphoma (including B-cell diffuse large cell lymphoma(DLCL), Diffuse Mixed Cell Lymphoma, Immunoblastic Lymphoma, PrimaryMediastinal B-Cell Lymphoma, Angiocentric Lymphoma-Pulmonary B-CellLymphoma), f) hairy cell leukemia, g) lymphocytic lymphoma,waldenstrom's macroglobulinemia, h) acute lymphocytic leukemia (ALL),chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL),B-cell prolymphocytic leukemia, i) plasma cell neoplasms, plasma cellmyeloma, multiple myeloma, plasmacytoma, j) Hodgkin's disease.

In one aspect, the CD20 expressing cancer is a B-Cell Non-Hodgkin'slymphoma (NHL). In another aspect, the CD20 expressing cancer isselected from the group consisting of Mantle cell lymphoma (MCL), acutelymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-celldiffuse large cell lymphoma (DLCL), Burkitt's lymphoma, hairy cellleukemia, follicular lymphoma, multiple myeloma, marginal zone lymphoma,post transplant lymphoproliferative disorder (PTLD), HIV associatedlymphoma, waldenstrom's macroglobulinemia, or primary CNS lymphoma.

By “B cell proliferative disorder” is meant a disease wherein the numberof B cells in a patient is increased as compared to the number of Bcells in a healthy subject, and particularly wherein the increase in thenumber of B cells is the cause or hallmark of the disease. A“CD20-positive B cell proliferative disorder” is a B cell proliferativedisorder wherein B-cells, particularly malignant B-cells (in addition tonormal B-cells), express CD20. Exemplary B cell proliferation disordersinclude Non-Hodgkin lymphoma (NHL), acute lymphocytic leukemia (ALL),chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma(DLBCL), follicular lymphoma (FL), mantle-cell lymphoma (MCL), marginalzone lymphoma (MZL), as well as some types of Multiple myeloma (MM) andHodgkin lymphoma (HL).

The term “a method of treating”, “a method of treatment” or itsequivalent, when applied to, for example, cancer refers to a procedureor course of action that is designed to reduce or eliminate the numberof cancer cells in a patient, or to alleviate the symptoms of a cancer.“A method of treating” cancer or another proliferative disorder does notnecessarily mean that the cancer cells or other disorder will, in fact,be eliminated, that the number of cells or disorder will, in fact, bereduced, or that the symptoms of a cancer or other disorder will, infact, be alleviated. Often, a method of treating cancer will beperformed even with a low likelihood of success, but which, given themedical history and estimated survival expectancy of a patient, isnevertheless deemed to induce an overall beneficial course of action.

The terms “combination”, “co-administration” or “co-administering” referto the administration of an anti-CD20/anti-CD3 bispecific antibody, andan anti-PD1/anti-LAG3 bispecific antibody as two separate formulations(or as one single formulation). The co-administration can besimultaneous or sequential in either order, wherein preferably there isa time period while both (or all) active agents simultaneously exerttheir biological activities. The anti-CD20/anti-CD3 bispecific antibodyand the anti-PD1/anti-LAG3 bispecific antibody are co-administeredeither simultaneously or sequentially (e.g. intravenous (i.v.) through acontinuous infusion (one for the anti-CD20/anti-CD3 bispecific antibodyand one for the anti-PD1/anti-LAG3 bispecific antibody). When boththerapeutic agents are co-administered sequentially, the dose isadministered either on the same day in two separate administrations, orone of the agents is administered on day 1 and the second isco-administered on day 2 to day 7, preferably on day 2 to 4. Thus theterm “sequentially” means within 7 days after the dose of the firstcomponent (anti-CD20/anti-CD3 bispecific antibody or theanti-PD1/anti-LAG3 bispecific antibody), preferably within 4 days afterthe dose of the first component; and the term “simultaneously” means atthe same time. The terms “co-administration” with respect to themaintenance doses of the anti-CD20/anti-CD3 bispecific antibody and theanti-PD1/anti-LAG3 bispecific antibody mean that the maintenance dosescan be either co-administered simultaneously, if the treatment cycle isappropriate for both drugs, e.g. every week. Or the anti-PD1/anti-LAG3bispecific antibody is administered e.g. every second week and theanti-CD20/anti-CD3 bispecific antibody is administered every third week.Or the maintenance doses are co-administered sequentially, either withinone or within several days.

Exemplary Anti-CD20/Anti-CD3 Bispecific Antibodies for Use in theInvention

The present invention relates to anti-CD20/anti-CD3 bispecificantibodies and their use in combination with anti-PD1/anti-LAG3bispecific antibodies, in particular to their use in a method fortreating or delaying progression of CD20-expressing cancer, moreparticularly for treating or delaying progression of B-cellproliferative disorders. The anti-CD20/anti-CD3 bispecific antibodies asused herein are bispecific antibodies comprising a first antigen bindingdomain that binds to CD3, and a second antigen binding domain that bindsto CD20. They are thus targeting CD20-expressing B cells.

Thus, the anti-CD20/anti-CD3 bispecific antibody as used hereincomprises a first antigen binding domain comprising a heavy chainvariable region (V_(H)CD3) and a light chain variable region (V_(L)CD3),and a second antigen binding domain comprising a heavy chain variableregion (V_(H)CD20) and a light chain variable region (V_(L)CD20).

In a particular aspect, the anti-CD20/anti-CD3 bispecific antibody foruse in the combination comprises a first antigen binding domaincomprising a heavy chain variable region (VHCD3) comprising CDR-H1sequence of SEQ ID NO:41, CDR-H2 sequence of SEQ ID NO:42, and CDR-H3sequence of SEQ ID NO:43; and/or a light chain variable region(V_(L)CD3) comprising CDR-L1 sequence of SEQ ID NO:44, CDR-L2 sequenceof SEQ ID NO:45, and CDR-L3 sequence of SEQ ID NO:46. More particularly,the anti-CD20/anti-CD3 bispecific comprises a first antigen bindingdomain comprising a heavy chain variable region (V_(H)CD3) that is atleast 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acidsequence of SEQ ID NO:47 and/or a light chain variable region (V_(L)CD3)that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the aminoacid sequence of SEQ ID NO:48. In a further aspect, theanti-CD20/anti-CD3 bispecific antibody comprises a heavy chain variableregion (VHCD3) comprising the amino acid sequence of SEQ ID NO:47 and/ora light chain variable region (V_(L)CD3) comprising the amino acidsequence of SEQ ID NO:48.

In one aspect, the antibody that specifically binds to CD3 is afull-length antibody. In one aspect, the antibody that specificallybinds to CD3 is an antibody of the human IgG class, particularly anantibody of the human IgG₁ class. In one aspect, the antibody thatspecifically binds to CD3 is an antibody fragment, particularly a Fabmolecule or a scFv molecule, more particularly a Fab molecule. In aparticular aspect, the antibody that specifically binds to CD3 is acrossover Fab molecule wherein the variable domains or the constantdomains of the Fab heavy and light chain are exchanged (i.e. replaced byeach other). In one aspect, the antibody that specifically binds to CD3is a humanized antibody.

In another aspect, the anti-CD20/anti-CD3 bispecific antibody comprisesa second antigen binding domain comprising a heavy chain variable region(V_(H)CD20) comprising CDR-H1 sequence of SEQ ID NO:49, CDR-H2 sequenceof SEQ ID NO:50, and CDR-H3 sequence of SEQ ID NO:51, and/or a lightchain variable region (V_(L)CD20) comprising CDR-L1 sequence of SEQ IDNO:52, CDR-L2 sequence of SEQ ID NO:53, and CDR-L3 sequence of SEQ IDNO:54. More particularly, the anti-CD20/anti-CD3 bispecific comprises asecond antigen binding domain comprising a heavy chain variable region(V_(H)CD20) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identicalto the amino acid sequence of SEQ ID NO:55 and/or a light chain variableregion (V_(L)CD20) that is at least 90%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence of SEQ ID NO:56. In a furtheraspect, the anti-CD20/anti-CD3 bispecific comprises a second antigenbinding domain comprising a heavy chain variable region (V_(H)CD20)comprising the amino acid sequence of SEQ ID NO:55 and/or a light chainvariable region (V_(L)CD20) comprising the amino acid sequence of SEQ IDNO:56.

In another particular aspect, the anti-CD20/anti-CD3 bispecific antibodycomprises a third antigen binding domain that binds to CD20. Inparticular, the anti-CD20/anti-CD3 bispecific antibody comprises a thirdantigen binding domain comprising a heavy chain variable region(V_(H)CD20) comprising CDR-H1 sequence of SEQ ID NO:49, CDR-H2 sequenceof SEQ ID NO:50, and CDR-H3 sequence of SEQ ID NO:51; and/or a lightchain variable region (V_(L)CD20) comprising CDR-L1 sequence of SEQ IDNO:52, CDR-L2 sequence of SEQ ID NO:53, and CDR-L3 sequence of SEQ IDNO:54. More particularly, the anti-CD20/anti-CD3 bispecific comprises athird antigen binding domain comprising a heavy chain variable region(V_(H)CD20) that is at least 90%, 95%, 96%, 97%, 98%, or 99% identicalto the amino acid sequence of SEQ ID NO:55 and/or a light chain variableregion (V_(L)CD20) that is at least 90%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence of SEQ ID NO:56. In a furtheraspect, the anti-CD20/anti-CD3 bispecific comprises a third antigenbinding domain comprising a heavy chain variable region (V_(H)CD20)comprising the amino acid sequence of SEQ ID NO:55 and/or a light chainvariable region (V_(L)CD20) comprising the amino acid sequence of SEQ IDNO:56.

In a further aspect, the anti-CD20/anti-CD3 bispecific antibody isbispecific antibody, wherein the first antigen binding domain is across-Fab molecule wherein the variable domains or the constant domainsof the Fab heavy and light chain are exchanged, and the second andthird, if present, antigen binding domain is a conventional Fabmolecule.

In another aspect, the anti-CD20/anti-CD3 bispecific antibody isbispecific antibody, wherein (i) the second antigen binding domain isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the first antigen binding domain, the first antigenbinding domain is fused at the C-terminus of the Fab heavy chain to theN-terminus of the first subunit of the Fc domain, and the third antigenbinding domain is fused at the C-terminus of the Fab heavy chain to theN-terminus of the second subunit of the Fc domain, or (ii) the firstantigen binding domain is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the second antigen bindingdomain, the second antigen binding domain is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the first subunit of the Fcdomain, and the third antigen binding domain is fused at the C-terminusof the Fab heavy chain to the N-terminus of the second subunit of the Fcdomain.

The Fab molecules may be fused to the Fc domain or to each otherdirectly or through a peptide linker, comprising one or more aminoacids, typically about 2-20 amino acids. Peptide linkers are known inthe art and are described herein. Suitable, non-immunogenic peptidelinkers include, for example, (G4S) (SEQ ID NO:71), (G₄S)₂ or GGGGSGGGGS(SEQ ID NO:72), (G4S)₃ (SEQ ID NO:73) and (G₄S)₄ (SEQ ID NO:74), moreparticularly (G₄S)₂ or GGGGSGGGGS (SEQ ID NO:72). A particularlysuitable peptide linker for fusing the Fab light chains of the first andthe second Fab molecule to each other is (G₄S)₂. Another suitable linkercomprises the sequence (G₄S)₄ (G₄S)₄ (SEQ ID NO:74). Additionally,linkers may comprise (a portion of) an immunoglobulin hinge region.Particularly where a Fab molecule is fused to the N-terminus of an Fcdomain subunit, it may be fused via an immunoglobulin hinge region or aportion thereof, with or without an additional peptide linker.

In a further aspect, the anti-CD20/anti-CD3 bispecific antibodycomprises an Fc domain comprising one or more amino acid substitutionsthat reduce binding to an Fc receptor and/or effector function. Inparticular, the anti-CD20/anti-CD3 bispecific antibody comprises an IgG1Fc domain comprising the amino acid substitutions L234A, L235A and P329G(according to EU numbering).

In a particular aspect, the anti-CD20/anti-CD3 bispecific antibodycomprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO: 57, a polypeptide that is atleast 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:58, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identicalto the sequence of SEQ ID NO: 59, and a polypeptide that is at least95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 60.In a further particular embodiment, the bispecific antibody comprises apolypeptide sequence of SEQ ID NO: 57, a polypeptide sequence of SEQ IDNO: 58, a polypeptide sequence of SEQ ID NO: 59 and a polypeptidesequence of SEQ ID NO: 60 (CD20 TCB).

In a particular aspect, the anti-CD20/anti-CD3 bispecific antibody isglofitamab.

Glofitamab (Proposed INN: List 121 WHO Drug Information, Vol. 33, No. 2,2019, also known as CD20-TCB, RO7082859, or RG6026) is a novelT-cell-engaging bispecific full-length antibody with a 2:1 molecularconfiguration for bivalent binding to CD20 on B cells and monovalentbinding to CD3, particularly the CD3 epsilon chain (CD3e), on T cells.Its CD3-binding region is fused to one of the CD20-binding regions in ahead-to-tail fashion via a flexible linker. This structure endowsglofitamab with superior in vitro potency versus other CD20-CD3bispecific antibodies with a 1:1 configuration, and leads to profoundantitumor efficacy in preclinical DLBCL models. CD20 bivalency preservesthis potency in the presence of competing anti-CD20 antibodies,providing the opportunity for pre- or co-treatment with these agents.Glofitamab comprises an engineered, heterodimeric Fc region withcompletely abolished binding to FcgRs and C1q. By simultaneously bindingto human CD20-expressing tumor cells and to the CD3ε of the T-cellreceptor (TCR) complex on T-cells, it induces tumor cell lysis, inaddition to T-cell activation, proliferation and cytokine release. Lysisof B-cells mediated by glofitamab is CD20-specific and does not occur inthe absence of CD20 expression or in the absence of simultaneous binding(cross-linking) of T-cells to CD20-expressing cells. In addition tokilling, T-cells undergo activation due to CD3 cross-linking, asdetected by an increase in T-cell activation markers (CD25 and CD69),cytokine release (IFNγ, TNFα, IL-2, IL-6, IL-10), cytotoxic granulerelease (Granzyme B) and T-cell proliferation.

In another aspect, the anti-CD20/anti-CD3 bispecific antibody for use inthe combination comprises a first antigen binding domain comprising aheavy chain variable region (V_(H)CD3) comprising CDR-H1 sequence of SEQID NO:83, CDR-H2 sequence of SEQ ID NO:84, and CDR-H3 sequence of SEQ IDNO:85; and/or a light chain variable region (V_(L)CD3) comprising CDR-L1sequence of SEQ ID NO:86, CDR-L2 sequence of SEQ ID NO:87, and CDR-L3sequence of SEQ ID NO:88. More particularly, the anti-CD20/anti-CD3bispecific comprises a first antigen binding domain comprising a heavychain variable region (VHCD3) that is at least 90%, 95%, 96%, 97%, 98%,or 99% identical to the amino acid sequence of SEQ ID NO:89 and/or alight chain variable region (V_(L)CD3) that is at least 90%, 95%, 96%,97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:90.In a further aspect, the anti-CD20/anti-CD3 bispecific antibodycomprises a heavy chain variable region (VHCD3) comprising the aminoacid sequence of SEQ ID NO:89 and/or a light chain variable region(V_(L)CD3) comprising the amino acid sequence of SEQ ID NO:90.

In a further aspect, the anti-CD20/anti-CD3 bispecific antibodycomprises a second antigen binding domain comprising a heavy chainvariable region (V_(H)CD20) comprising CDR-H1 sequence of SEQ ID NO:91,CDR-H2 sequence of SEQ ID NO:92, and CDR-H3 sequence of SEQ ID NO:93,and/or a light chain variable region (V_(L)CD20) comprising CDR-L1sequence of SEQ ID NO:94, CDR-L2 sequence of SEQ ID NO:95, and CDR-L3sequence of SEQ ID NO:96. More particularly, the anti-CD20/anti-CD3bispecific comprises a second antigen binding domain comprising a heavychain variable region (V_(H)CD20) that is at least 90%, 95%, 96%, 97%,98%, or 99% identical to the amino acid sequence of SEQ ID NO:97 and/ora light chain variable region (V_(L)CD20) that is at least 90%, 95%,96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ IDNO:98. In a further aspect, the anti-CD20/anti-CD3 bispecific comprisesa second antigen binding domain comprising a heavy chain variable region(V_(H)CD20) comprising the amino acid sequence of SEQ ID NO:97 and/or alight chain variable region (V_(L)CD20) comprising the amino acidsequence of SEQ ID NO:98.

In a particular aspect, the anti-CD20/anti-CD3 bispecific antibodycomprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO: 99, a polypeptide that is atleast 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:100, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identicalto the sequence of SEQ ID NO: 101, and a polypeptide that is at least95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 102.In a further particular embodiment, the bispecific antibody comprises apolypeptide sequence of SEQ ID NO: 99, a polypeptide sequence of SEQ IDNO: 100, a polypeptide sequence of SEQ ID NO: 101 and a polypeptidesequence of SEQ ID NO: 102.

In a particular aspect, the anti-CD20/anti-CD3 bispecific antibody ismosunetuzumab.—Mosunetuzumab (R07030816; also known as BTCT4465A) is ahumanized full-length anti-CD20/CD3 T-cell dependent bispecific (TDB)antibody of the human IgG1 class comprising an amino acid substitutionN297G (according to EU Numbering) in the fragment crystallizable (Fc)region. This substitution results in a non-glycosylated heavy chain thathas minimal binding to Fc gamma (FC-γ) receptors and, consequently,reduces Fc effector functions. The mechanism of action of mosunetuzumabinvolves engaging T-cells via CD3 with CD20-expressing cells, leading toT-cell activation and T-cell mediated cytolysis of the CD20-expressingcells. On the basis of its structure as a full-length antibody andnonclinical data, the pharmacokinetic (PK) properties of mosunetuzumabenable intermittent dosing in the clinical setting, similar to othermonoclonal antibodies.

Particular bispecific antibodies are described in PCT publication no. WO2016/020309 A1 or in WO 2015/095392 A1.

In a further aspect, the anti-CD20/anti-CD3 bispecific antibody may alsocomprise a bispecific T cell engager (BiTE®). In a further aspect, theanti-CD20/anti-CD3 bispecific antibody is XmAb®13676. In another aspect,the bispecific antibody is REGN1979. In another aspect, the bispecificantibody is FBTA05 (Lymphomun).

Exemplary Bispecific Anti-PD1/Anti-LAG3 Antibodies for Use in theInvention

For the combination provided herein, used are novel bispecificantibodies comprising a first antigen binding domain that specificallybinds to programmed cell death protein 1 (PD1) and a second antigenbinding domain that specifically binds to Lymphocyte activation gene-3(LAG3), with particularly advantageous properties such as producibility,stability, binding affinity, biological activity, specific targeting ofcertain T cells, targeting efficiency and reduced toxicity. Particularbispecific anti-PD1/anti-LAG3 antibodies for use herein are described inWO 2018/185043 A1.

In certain aspects, a bispecific antibody comprising a first antigenbinding domain that specifically binds to PD1 and a second antigenbinding domain that specifically binds to LAG3 is provided that showsreduced internalization upon binding to the T cell surface. Theinternalization represents an important sink for the molecule which canbe degraded within a few hours while the targeted receptors are rapidlyre-expressed on the cell-surface ready to inhibit TCR-signalling. Infurther aspects, a bispecific antibody comprising a first antigenbinding domain that specifically binds to PD1 and a second antigenbinding domain that specifically binds to LAG3 is provided thatpreferentially binds to conventional T cells rather than to Tregs. Thisis advantageous because targeting LAG-3 on Tregs with blockingantibodies could be detrimental by increasing their suppressive functionand eventually mask the positive blocking effect on other T cells. In afurther aspect, a bispecific antibody comprising a first antigen bindingdomain that specifically binds to PD1 and a second antigen bindingdomain that specifically binds to LAG3 is provided that is able torescue T cell effector functions from Treg suppression. In anotheraspect, a bispecific antibody comprising a first antigen binding domainthat specifically binds to PD1 and a second antigen binding domain thatspecifically binds to LAG3 is provided that is able to induce Granzyme Bsecretion by CD4 T cells, when co-cultured with the tumor cell lineARH77 as shown in the assay provided herein. In a further aspect, abispecific antibody comprising a first antigen binding domain thatspecifically binds to PD1 and a second antigen binding domain thatspecifically binds to LAG3 is provided that shows increasedtumor-specific T cell effector functions and/or enhances the cytotoxiceffect of T cells. In another aspect, a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3 is provided thatshows increased tumor eradication in vivo.

In one aspect, the invention provides a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein saidfirst antigen binding domain specifically binding to PD1 comprises

-   -   a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:            1,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:2, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:3; and    -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:4;        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:            5, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO: 6.

In one aspect, the bispecific antibody comprises a Fc domain that is anIgG, particularly an IgG1 Fc domain or an IgG4 Fc domain and wherein theFc domain has reduced or even abolished effector function. Inparticular, the Fc domain comprises one or more amino acid substitutionthat reduces binding to an Fc receptor, in particular towards Fcγreceptor.

In a further aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thebispecific antibody comprises a Fc domain that is an IgG, particularlyan IgG1 Fc domain or an IgG4 Fc domain and wherein the Fc domaincomprises one or more amino acid substitution that reduces binding to anFc receptor, in particular towards Fcγ receptor.

In another aspect, provided is a bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thesecond antigen binding domain that specifically binds to LAG3 comprises

-   -   (a) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:11,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:            12, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:13; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:14,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:15, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO: 16; or    -   (b) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:19,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:20, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:21; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:22,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:23, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:24.

In a further aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thefirst antigen binding domain specifically binding to PD1 comprises theVH domain comprising the amino acid sequence of SEQ ID NO: 9 and the VLdomain comprising the amino acid sequence of SEQ ID NO:10.

In another aspect, provided is a bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thesecond antigen binding domain specifically binding to LAG3 comprises

-   -   (a) a VH domain comprising the amino acid sequence of SEQ ID        NO:17 and a VL domain comprising the amino acid sequence of SEQ        ID NO:18, or    -   (b) a VH domain comprising the amino acid sequence of SEQ ID NO:        25 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 26.

In a further aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thesecond antigen binding domain specifically binding to LAG3 comprises

-   -   (a) a VH domain comprising the amino acid sequence of SEQ ID NO:        27 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 28, or    -   (b) a VH domain comprising the amino acid sequence of SEQ ID NO:        29 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 30, or    -   (c) a VH domain comprising the amino acid sequence of SEQ ID NO:        31 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 32, or    -   (d) a VH domain comprising the amino acid sequence of SEQ ID NO:        33 and a VL domain comprising the amino acid sequence of SEQ ID        NO: 34.

In another aspect, provided is a bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thesecond antigen binding domain specifically binding to LAG3 comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 81 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 82.

In a particular aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein

-   -   the first antigen binding domain specifically binding to PD1        comprises a VH domain comprising the amino acid sequence of SEQ        ID NO: 9 and a VL domain comprising the amino acid sequence of        SEQ ID NO: 10,    -   and the second antigen binding domain specifically binding to        LAG3 comprises a VH domain comprising the amino acid sequence of        SEQ ID NO: 17 and a VL domain comprising the amino acid sequence        of SEQ ID NO: 18 or a VH domain comprising the amino acid        sequence of SEQ ID NO: 25 and a VL domain comprising the amino        acid sequence of SEQ ID NO: 26.

In one aspect, the bispecific antibody of the invention comprises afirst antigen binding domain specifically binding to PD1 comprising a VHdomain comprising the amino acid sequence of SEQ ID NO: 9 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 10 and a secondantigen binding domain specifically binding to LAG3 comprising a VHdomain comprising the amino acid sequence of SEQ ID NO: 17 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 18.

In a further aspect, the bispecific antibody of the invention comprisesa first antigen binding domain specifically binding to PD1 comprising aVH domain comprising the amino acid sequence of SEQ ID NO: 9 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 10 and a secondantigen binding domain specifically binding to LAG3 comprising a VHdomain comprising the amino acid sequence of SEQ ID NO: 25 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 26.

In a further aspect, the bispecific antibody comprising a first antigenbinding domain that specifically binds to PD1 and a second antigenbinding domain that specifically binds to LAG3 is a human, humanized orchimeric antibody. In particular, it is a humanized or chimericantibody.

In one aspect, the bispecific antibody comprising a first antigenbinding domain that specifically binds to PD1 and a second antigenbinding domain that specifically binds to LAG3 is bivalent. This meansthat the bispecific antibody comprises one antigen binding domain thatspecifically binds to PD1 and one antigen binding domain thatspecifically binds to LAG3 (1+1 format).

In one aspect, provided is a bispecific antibody comprising a firstantigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thebispecific antibody comprises an Fc domain, a first Fab fragmentcomprising the antigen binding domain that specifically binds to PD1 anda second Fab fragment comprising the antigen binding domain thatspecifically binds to LAG3. In a particular aspect, in one of the Fabfragments the variable domains VL and VH are replaced by each other sothat the VH domain is part of the light chain and the VL domain is partof the heavy chain. In a particular aspect, in the first Fab fragmentcomprising the antigen binding domain that specifically binds to PD1 thevariable domains VL and VH are replaced by each other.

In a particular aspect, provided is a bispecific antibody comprising afirst antigen binding domain that specifically binds to PD1 and a secondantigen binding domain that specifically binds to LAG3, wherein thebispecific antibody comprises

-   -   (a) a first heavy chain comprising an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO: 35,        a first light chain comprising an amino acid sequence with at        least 95% sequence identity to the sequence of SEQ ID NO: 36, a        second heavy chain comprising an amino acid sequence with at        least 95% sequence identity to the sequence of SEQ ID NO: 37,        and a second light chain comprising an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO:38,        or    -   (b) a first heavy chain comprising an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO: 35,        a first light chain comprising an amino acid sequence with at        least 95% sequence identity to the sequence of SEQ ID NO: 36, a        second heavy chain comprising an amino acid sequence with at        least 95% sequence identity to the sequence of SEQ ID NO: 39,        and a second light chain comprising an amino acid sequence with        at least 95% sequence identity to the sequence of SEQ ID NO:40.

More particularly, the bispecific antibody comprises a first heavy chaincomprising an amino acid sequence of SEQ ID NO: 35, a first light chaincomprising an amino acid sequence of SEQ ID NO: 36, a second heavy chaincomprising an amino acid sequence of SEQ ID NO: 37, and a second lightchain comprising an amino acid sequence of SEQ ID NO:38.

Fc Domain Modifications Reducing Fc Receptor Binding and/or EffectorFunction

In certain aspects, provided is an anti-PD1/anti-LAG3 bispecificantibody, wherein the bispecific antibody comprises a Fc domaincomprising one or more amino acid modifications that reduce binding toan Fc receptor, in particular towards Fcγ receptor, and reduce orabolish effector function.

In certain aspects, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

The following section describes preferred aspects of the bispecificantigen binding molecules of the invention comprising Fc domainmodifications reducing Fc receptor binding and/or effector function. Inone aspect, the invention relates to an anti-PD1/anti-LAG3 bispecificantibody, wherein the Fc domain comprises one or more amino acidsubstitution that reduces binding to an Fc receptor, in particulartowards Fcγ receptor. In particular, the Fc domain is of human IgG1subclass with the amino acid mutations L234A, L235A and P329G (numberingaccording to Kabat EU index).

The Fc domain confers favorable pharmacokinetic properties to thebispecific antibodies of the invention, including a long serum half-lifewhich contributes to good accumulation in the target tissue and afavorable tissue-blood distribution ratio. At the same time it may,however, lead to undesirable targeting of the bispecific antibodies ofthe invention to cells expressing Fc receptors rather than to thepreferred antigen-bearing cells. Accordingly, in particular embodimentsthe Fc domain of the bispecific antibodies of the invention exhibitsreduced binding affinity to an Fc receptor and/or reduced effectorfunction, as compared to a native IgG Fc domain, in particular an IgG1Fc domain or an IgG4 Fc domain. More particularly, the Fe domain is anIgG1 FC domain.

In one such aspect the Fc domain (or the bispecific antigen bindingmolecule of the invention comprising said Fc domain) exhibits less than50%, preferably less than 20%, more preferably less than 10% and mostpreferably less than 5% of the binding affinity to an Fc receptor, ascompared to a native IgG1 Fc domain (or the bispecific antigen bindingmolecule of the invention comprising a native IgG1 Fc domain), and/orless than 50%, preferably less than 20%, more preferably less than 10%and most preferably less than 5% of the effector function, as comparedto a native IgG1 Fc domain (or the bispecific antigen binding moleculeof the invention comprising a native IgG1 Fc domain). In one aspect, theFc domain (or the bispecific antigen binding molecule of the inventioncomprising said Fc domain) does not substantially bind to an Fc receptorand/or induce effector function. In a particular aspect the Fc receptoris an Fcγ receptor. In one aspect, the Fc receptor is a human Fcreceptor. In one aspect, the Fc receptor is an activating Fc receptor.In a specific aspect, the Fc receptor is an activating human Fcγreceptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, mostspecifically human FcγRIIIa. In one aspect, the Fc receptor is aninhibitory Fc receptor. In a specific aspect, the Fc receptor is aninhibitory human Fcγ receptor, more specifically human FcγRIIB. In oneaspect the effector function is one or more of CDC, ADCC, ADCP, andcytokine secretion. In a particular aspect, the effector function isADCC. In one aspect, the Fc domain exhibits substantially similarbinding affinity to neonatal Fc receptor (FcRn), as compared to a nativeIgG1 Fc domain. Substantially similar binding to FcRn is achieved whenthe Fc domain (or the bispecific antigen binding molecule of theinvention comprising said Fc domain) exhibits greater than about 70%,particularly greater than about 80%, more particularly greater thanabout 90% of the binding affinity of a native IgG1 Fc domain (or thebispecific antigen binding molecule of the invention comprising a nativeIgG1 Fc domain) to FcRn.

In a particular aspect, the Fc domain is engineered to have reducedbinding affinity to an Fc receptor and/or reduced effector function, ascompared to a non-engineered Fc domain. In a particular aspect, the Fcdomain of the bispecific antigen binding molecule of the inventioncomprises one or more amino acid mutation that reduces the bindingaffinity of the Fc domain to an Fc receptor and/or effector function.Typically, the same one or more amino acid mutation is present in eachof the two subunits of the Fc domain. In one aspect, the amino acidmutation reduces the binding affinity of the Fc domain to an Fcreceptor. In another aspect, the amino acid mutation reduces the bindingaffinity of the Fc domain to an Fc receptor by at least 2-fold, at least5-fold, or at least 10-fold. In one aspect, the bispecific antigenbinding molecule of the invention comprising an engineered Fc domainexhibits less than 20%, particularly less than 10%, more particularlyless than 5% of the binding affinity to an Fc receptor as compared tobispecific antibodies of the invention comprising a non-engineered Fcdomain. In a particular aspect, the Fc receptor is an Fcγ receptor. Inother aspects, the Fc receptor is a human Fc receptor. In one aspect,the Fc receptor is an inhibitory Fc receptor. In a specific aspect, theFc receptor is an inhibitory human Fcγ receptor, more specifically humanFcγRIIB. In some aspects the Fc receptor is an activating Fc receptor.In a specific aspect, the Fc receptor is an activating human Fcγreceptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, mostspecifically human FcγRIIIa. Preferably, binding to each of thesereceptors is reduced. In some aspects, binding affinity to a complementcomponent, specifically binding affinity to C1q, is also reduced. In oneaspect, binding affinity to neonatal Fc receptor (FcRn) is not reduced.Substantially similar binding to FcRn, i.e. preservation of the bindingaffinity of the Fc domain to said receptor, is achieved when the Fcdomain (or the bispecific antigen binding molecule of the inventioncomprising said Fc domain) exhibits greater than about 70% of thebinding affinity of a non-engineered form of the Fc domain (or thebispecific antigen binding molecule of the invention comprising saidnon-engineered form of the Fc domain) to FcRn. The Fc domain, or thebispecific antigen binding molecule of the invention comprising said Fcdomain, may exhibit greater than about 80% and even greater than about90% of such affinity. In certain embodiments the Fc domain of thebispecific antigen binding molecule of the invention is engineered tohave reduced effector function, as compared to a non-engineered Fcdomain. The reduced effector function can include, but is not limitedto, one or more of the following: reduced complement dependentcytotoxicity (CDC), reduced antibody-dependent cell-mediatedcytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis(ADCP), reduced cytokine secretion, reduced immune complex-mediatedantigen uptake by antigen-presenting cells, reduced binding to NK cells,reduced binding to macrophages, reduced binding to monocytes, reducedbinding to polymorphonuclear cells, reduced direct signaling inducingapoptosis, reduced dendritic cell maturation, or reduced T cell priming.

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581). Certain antibody variants with improved or diminishedbinding to FcRs are described. (e.g. U.S. Pat. No. 6,737,056; WO2004/056312, and Shields, R. L. et al., J. Biol. Chem. 276 (2001)6591-6604).

In one aspect of the invention, the Fc domain comprises an amino acidsubstitution at a position of E233, L234, L235, N297, P331 and P329. Insome aspects, the Fc domain comprises the amino acid substitutions L234Aand L235A (“LALA”). In one such embodiment, the Fc domain is an IgG1 Fcdomain, particularly a human IgG1 Fc domain. In one aspect, the Fcdomain comprises an amino acid substitution at position P329. In a morespecific aspect, the amino acid substitution is P329A or P329G,particularly P329G. In one embodiment the Fc domain comprises an aminoacid substitution at position P329 and a further amino acid substitutionselected from the group consisting of E233P, L234A, L235A, L235E, N297A,N297D or P331S. In more particular embodiments the Fc domain comprisesthe amino acid mutations L234A, L235A and P329G (“P329G LALA”). The“P329G LALA” combination of amino acid substitutions almost completelyabolishes Fcγ receptor binding of a human IgG1 Fc domain, as describedin PCT Patent Application No. WO 2012/130831 A1. Said document alsodescribes methods of preparing such mutant Fc domains and methods fordetermining its properties such as Fc receptor binding or effectorfunctions. such antibody is an IgG1 with mutations L234A and L235A orwith mutations L234A, L235A and P329G (numbering according to EU indexof Kabat et al, Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, M D, 1991).

In one aspect, the anti-PD1/anti-LAG3 bispecific antibody comprises (allpositions according to EU index of Kabat) (i) a homodimeric Fc-region ofthe human IgG1 subclass optionally with the mutations P329G, L234A andL235A, or (ii) a homodimeric Fc-region of the human IgG4 subclassoptionally with the mutations P329G, S228P and L235E, or (iii) ahomodimeric Fc-region of the human IgG1 subclass optionally with themutations P329G, L234A, L235A, 1253A, H310A, and H435A, or optionallywith the mutations P329G, L234A, L235A, H310A, H433A, and Y436A, or (iv)a heterodimeric Fc-region wherein one Fc-region polypeptide comprisesthe mutation T366W, and the other Fc-region polypeptide comprises themutations T366S, L368A and Y407V, or wherein one Fc-region polypeptidecomprises the mutations T366W and Y349C, and the other Fc-regionpolypeptide comprises the mutations T366S, L368A, Y407V, and S354C, orwherein one Fc-region polypeptide comprises the mutations T366W andS354C, and the other Fc-region polypeptide comprises the mutationsT366S, L368A, Y407V and Y349C, or (v) a heterodimeric Fc-region of thehuman IgG1 subclass wherein both Fc-region polypeptides comprise themutations P329G, L234A and L235A and one Fc-region polypeptide comprisesthe mutation T366W, and the other Fc-region polypeptide comprises themutations T366S, L368A and Y407V, or wherein one Fc-region polypeptidecomprises the mutations T366W and Y349C, and the other Fc-regionpolypeptide comprises the mutations T366S, L368A, Y407V, and S354C, orwherein one Fc-region polypeptide comprises the mutations T366W andS354C, and the other Fc-region polypeptide comprises the mutationsT366S, L368A, Y407V and Y349C.

In one aspect, the Fc domain is an IgG4 Fc domain. In a more specificembodiment, the Fc domain is an IgG4 Fc domain comprising an amino acidsubstitution at position S228 (Kabat numbering), particularly the aminoacid substitution S228P. In a more specific embodiment, the Fc domain isan IgG4 Fc domain comprising amino acid substitutions L235E and S228Pand P329G. This amino acid substitution reduces in vivo Fab arm exchangeof IgG4 antibodies (see Stubenrauch et al., Drug Metabolism andDisposition 38, 84-91 (2010)). Thus, in one aspect, provided is abispecific antibody, comprising (all positions according to EU index ofKabat) a heterodimeric Fc-region of the human IgG4 subclass wherein bothFc-region polypeptides comprise the mutations P329G, S228P and L235E andone Fc-region polypeptide comprises the mutation T366W, and the otherFc-region polypeptide comprises the mutations T366S, L368A and Y407V, orwherein one Fc-region polypeptide comprises the mutations T366W andY349C, and the other Fc-region polypeptide comprises the mutationsT366S, L368A, Y407V, and S354C, or wherein one Fc-region polypeptidecomprises the mutations T366W and S354C, and the other Fc-regionpolypeptide comprises the mutations T366S, L368A, Y407V and Y349C.

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer, R. L. et al., J. Immunol. 117 (1976)587-593, and Kim, J. K. et al., J. Immunol. 24 (1994) 2429-2434), aredescribed in US 2005/0014934. Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).See also Duncan, A. R. and Winter, G., Nature 322 (1988) 738-740; U.S.Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351 concerning otherexamples of Fc region variants.

Binding to Fc receptors can be easily determined e.g. by ELISA, or bySurface Plasmon Resonance (SPR) using standard instrumentation such as aBIAcore instrument (GE Healthcare), and Fc receptors such as may beobtained by recombinant expression. A suitable such binding assay isdescribed herein. Alternatively, binding affinity of Fc domains or cellactivating bispecific antigen binding molecules comprising an Fc domainfor Fc receptors may be evaluated using cell lines known to expressparticular Fc receptors, such as human NK cells expressing FcγIIIareceptor. Effector function of an Fc domain, or bispecific antibodies ofthe invention comprising an Fc domain, can be measured by methods knownin the art. A suitable assay for measuring ADCC is described herein.Other examples of in vitro assays to assess ADCC activity of a moleculeof interest are described in U.S. Pat. No. 5,500,362; Hellstrom et al.Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., ProcNatl Acad Sci USA 82, 1499-1502 (1985); U.S. Pat. No. 5,821,337;Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively,non-radioactive assays methods may be employed (see, for example, ACTI™non-radioactive cytotoxicity assay for flow cytometry (CellTechnology,Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicityassay (Promega, Madison, WI)). Useful effector cells for such assaysinclude peripheral blood mononuclear cells (PBMC) and Natural Killer(NK) cells. Alternatively, or additionally, ADCC activity of themolecule of interest may be assessed in vivo, e.g. in a animal modelsuch as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95,652-656 (1998).

The following section describes preferred aspects of the bispecificantibodies of the invention comprising Fc domain modifications reducingFc receptor binding and/or effector function. In one aspect, provided isan anti-PD1/anti-LAG3 bispecific antibody, wherein the Fc domaincomprises one or more amino acid substitution that reduces the bindingaffinity of the antibody to an Fc receptor, in particular towards Fcγreceptor. In another aspect, provided is an anti-PD1/anti-LAG3bispecific antibody, wherein the Fc domain comprises one or more aminoacid substitution that reduces effector function. In particular aspect,the Fc domain is of human IgG1 subclass with the amino acid mutationsL234A, L235A and P329G (numbering according to Kabat EU index).

Fc Domain Modifications Promoting Heterodimerization

The bispecific antigen binding molecules as described herein comprisedifferent antigen binding domains, fused to one or the other of the twosubunits of the Fc domain, thus the two subunits of the Fc domain may becomprised in two non-identical polypeptide chains. Recombinantco-expression of these polypeptides and subsequent dimerization leads toseveral possible combinations of the two polypeptides. To improve theyield and purity of the bispecific antibodies of the invention inrecombinant production, it will thus be advantageous to introduce in theFc domain of the bispecific antigen binding molecules described herein amodification promoting the association of the desired polypeptides.

Accordingly, in particular aspects provided is an anti-PD1/anti-LAG3bispecific antibody, wherein the Fc domain comprises a modificationpromoting the association of the first and second subunit of the Fcdomain. The site of most extensive protein-protein interaction betweenthe two subunits of a human IgG Fc domain is in the CH3 domain of the Fcdomain. Thus, in one aspect said modification is in the CH3 domain ofthe Fc domain.

In a specific aspect said modification is a so-called “knob-into-hole”modification, comprising a “knob” modification in one of the twosubunits of the Fc domain and a “hole” modification in the other one ofthe two subunits of the Fc domain. Thus, the invention relates to abispecific antibody comprising a first antigen binding domain thatspecifically binds to PD1 and a second antigen-binding site thatspecifically binds to LAG3, wherein the first subunit of the Fc domaincomprises knobs and the second subunit of the Fc domain comprises holesaccording to the knobs into holes method. In a particular aspect, thefirst subunit of the Fc domain comprises the amino acid substitutionsS354C and T366W (EU numbering) and the second subunit of the Fc domaincomprises the amino acid substitutions Y349C, T366S and Y407V (numberingaccording to Kabat EU index).

The knob-into-hole technology is described e.g. in U.S. Pat. Nos.5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) andCarter, J Immunol Meth 248, 7-15 (2001). Generally, the method involvesintroducing a protuberance (“knob”) at the interface of a firstpolypeptide and a corresponding cavity (“hole”) in the interface of asecond polypeptide, such that the protuberance can be positioned in thecavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory cavities of identicalor similar size to the protuberances are created in the interface of thesecond polypeptide by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine).

Accordingly, in one aspect, in the CH3 domain of the first subunit ofthe Fc domain of the bispecific antigen binding molecules of theinvention an amino acid residue is replaced with an amino acid residuehaving a larger side chain volume, thereby generating a protuberancewithin the CH3 domain of the first subunit which is positionable in acavity within the CH3 domain of the second subunit, and in the CH3domain of the second subunit of the Fc domain an amino acid residue isreplaced with an amino acid residue having a smaller side chain volume,thereby generating a cavity within the CH3 domain of the second subunitwithin which the protuberance within the CH3 domain of the first subunitis positionable. The protuberance and cavity can be made by altering thenucleic acid encoding the polypeptides, e.g. by site-specificmutagenesis, or by peptide synthesis. In a specific aspect, in the CH3domain of the first subunit of the Fc domain the threonine residue atposition 366 is replaced with a tryptophan residue (T366W), and in theCH3 domain of the second subunit of the Fc domain the tyrosine residueat position 407 is replaced with a valine residue (Y407V). In oneaspect, in the second subunit of the Fc domain additionally thethreonine residue at position 366 is replaced with a serine residue(T366S) and the leucine residue at position 368 is replaced with analanine residue (L368A).

In yet a further aspect, in the first subunit of the Fc domainadditionally the serine residue at position 354 is replaced with acysteine residue (S354C), and in the second subunit of the Fc domainadditionally the tyrosine residue at position 349 is replaced by acysteine residue (Y349C). Introduction of these two cysteine residuesleads to the formation of a disulfide bridge between the two subunits ofthe Fc domain, further stabilizing the dimer (Carter (2001), J ImmunolMethods 248, 7-15). In a particular aspect, the first subunit of the Fcdomain comprises the amino acid substitutions S354C and T366W (EUnumbering) and the second subunit of the Fc domain comprises the aminoacid substitutions Y349C, T366S and Y407V (numbering according to KabatEU index).

But also other knobs-in-holes technologies as described by EP 1 870 459,can be used alternatively or additionally. In one embodiment themultispecific antibody comprises the mutations R409D and K370E in theCH3 domain of the “knobs chain” and the mutations D399K and E357K in theCH3 domain of the “hole-chain” (numbering according to Kabat EU index).

In one aspect, the bispecific antibody comprises a T366W mutation in theCH3 domain of the “knobs chain” and the mutations T366S, L368A and Y407Vin the CH3 domain of the “hole chain” and additionally the mutationsR409D and K370E in the CH3 domain of the “knobs chain” and the mutationsD399K and E357K in the CH3 domain of the “hole chain” (numberingaccording to the Kabat EU index).

In one aspect, the bispecific antibody comprises the mutations Y349C andT366W in one of the two CH3 domains and the mutations S354C, T366S,L368A and Y407V in the other of the two CH3 domains, or themultispecific antibody comprises the mutations Y349C and T366W in one ofthe two CH3 domains and the mutations S354C, T366S, L368A and Y407V inthe other of the two CH3 domains and additionally the mutations R409Dand K370E in the CH3 domain of the “knobs chain” and the mutations D399Kand E357K in the CH3 domain of the “hole chain” (numbering according tothe Kabat EU index).

In an alternative aspect, a modification promoting association of thefirst and the second subunit of the Fc domain comprises a modificationmediating electrostatic steering effects, e.g. as described in PCTpublication WO 2009/089004. Generally, this method involves replacementof one or more amino acid residues at the interface of the two Fc domainsubunits by charged amino acid residues so that homodimer formationbecomes electrostatically unfavorable but heterodimerizationelectrostatically favorable.

Apart from the “knob-into-hole technology” other techniques formodifying the CH3 domains of the heavy chains of a multispecificantibody to enforce heterodimerization are known in the art. Thesetechnologies, especially the ones described in WO 96/27011, WO98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004,WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO2013/157954 and WO 2013/096291 are contemplated herein as alternativesto the “knob-into-hole technology” in combination with a bispecificantibody.

In one aspect, in the bispecific antibody the approach described in EP1870459 is used to support heterodimerization of the first heavy chainand the second heavy chain of the multispecific antibody. This approachis based on the introduction of charged amino acids with oppositecharges at specific amino acid positions in the CH3/CH3-domain-interfacebetween both, the first and the second heavy chain.

Accordingly, in this aspect in the tertiary structure of themultispecific antibody the CH3 domain of the first heavy chain and theCH3 domain of the second heavy chain form an interface that is locatedbetween the respective antibody CH3 domains, wherein the respectiveamino acid sequences of the CH3 domain of the first heavy chain and theamino acid sequence of the CH3 domain of the second heavy chain eachcomprise a set of amino acids that is located within said interface inthe tertiary structure of the antibody, wherein from the set of aminoacids that is located in the interface in the CH3 domain of one heavychain a first amino acid is substituted by a positively charged aminoacid and from the set of amino acids that is located in the interface inthe CH3 domain of the other heavy chain a second amino acid issubstituted by a negatively charged amino acid. The bispecific antibodyaccording to this aspect is herein also referred to as“CH3(+/−)-engineered bispecific antibody” (wherein the abbreviation“+/−” stands for the oppositely charged amino acids that were introducedin the respective CH3 domains).

In one aspect, in the CH3(+/−)-engineered bispecific antibody thepositively charged amino acid is selected from K, R and H, and thenegatively charged amino acid is selected from E or D.

In one aspect, in the CH3(+/−)-engineered bispecific antibody thepositively charged amino acid is selected from K and R, and thenegatively charged amino acid is selected from E or D.

In one aspect, in the CH3(+/−)-engineered bispecific antibody thepositively charged amino acid is K, and the negatively charged aminoacid is E.

In one aspect, in the CH3(+/−)-engineered bispecific antibody in the CH3domain of one heavy chain the amino acid R at position 409 issubstituted by D and the amino acid K at position is substituted by E,and in the CH3 domain of the other heavy chain the amino acid D atposition 399 is substituted by K and the amino acid E at position 357 issubstituted by K (numbering according to Kabat EU index).

In one aspect, the approach described in WO 2013/157953 is used tosupport heterodimerization of the first heavy chain and the second heavychain of the multispecific antibody. In one embodiment in the CH3 domainof one heavy chain the amino acid T at position 366 is substituted by K,and in the CH3 domain of the other heavy chain the amino acid L atposition 351 is substituted by D (numbering according to Kabat EUindex). In another embodiment in the CH3 domain of one heavy chain theamino acid T at position 366 is substituted by K and the amino acid L atposition 351 is substituted by K, and in the CH3 domain of the otherheavy chain the amino acid L at position 351 is substituted by D(numbering according to Kabat EU index).

In another aspect, in the CH3 domain of one heavy chain the amino acid Tat position 366 is substituted by K and the amino acid L at position 351is substituted by K, and in the CH3 domain of the other heavy chain theamino acid L at position 351 is substituted by D (numbering according toKabat EU index). Additionally at least one of the followingsubstitutions is comprised in the CH3 domain of the other heavy chain:the amino acid Y at position 349 is substituted by E, the amino acid Yat position 349 is substituted by D and the amino acid L at position 368is substituted by E (numbering according to Kabat EU index). In oneembodiment the amino acid L at position 368 is substituted by E(numbering according to Kabat EU index).

In one aspect, the approach described in WO 2012/058768 is used tosupport heterodimerization of the first heavy chain and the second heavychain of the multispecific antibody. In one aspect, in the CH3 domain ofone heavy chain the amino acid L at position 351 is substituted by Y andthe amino acid Y at position 407 is substituted by A, and in the CH3domain of the other heavy chain the amino acid T at position 366 issubstituted by A and the amino acid K at position 409 is substituted byF (numbering according to Kabat EU index). In another embodiment, inaddition to the aforementioned substitutions, in the CH3 domain of theother heavy chain at least one of the amino acids at positions 411(originally T), 399 (originally D), 400 (originally S), 405 (originallyF), 390 (originally N) and 392 (originally K) is substituted (numberingaccording to Kabat EU index). Preferred substitutions are:

-   -   substituting the amino acid T at position 411 by an amino acid        selected from N, R, Q, K, D, E and W (numbering according to        Kabat EU index),    -   substituting the amino acid D at position 399 by an amino acid        selected from R, W, Y, and K (numbering according to Kabat EU        index),    -   substituting the amino acid S at position 400 by an amino acid        selected from E, D, R and K (numbering according to Kabat EU        index),    -   substituting the amino acid F at position 405 by an amino acid        selected from I, M, T, S, V and W (numbering according to Kabat        EU index;    -   substituting the amino acid N at position 390 by an amino acid        selected from R, K and D (numbering according to Kabat EU index;        and    -   substituting the amino acid K at position 392 by an amino acid        selected from V, M, R, L, F and E (numbering according to Kabat        EU index).

In another aspect, the bispecific antibody is engineered according to WO2012/058768), i.e. in the CH3 domain of one heavy chain the amino acid Lat position 351 is substituted by Y and the amino acid Y at position 407is substituted by A, and in the CH3 domain of the other heavy chain theamino acid T at position 366 is substituted by V and the amino acid K atposition 409 is substituted by F (numbering according to Kabat EUindex). In another embodiment of the multispecific antibody, in the CH3domain of one heavy chain the amino acid Y at position 407 issubstituted by A, and in the CH3 domain of the other heavy chain theamino acid T at position 366 is substituted by A and the amino acid K atposition 409 is substituted by F (numbering according to Kabat EUindex). In the last aforementioned embodiment, in the CH3 domain of theother heavy chain the amino acid K at position 392 is substituted by E,the amino acid T at position 411 is substituted by E, the amino acid Dat position 399 is substituted by R and the amino acid S at position 400is substituted by R (numbering according to Kabat EU index).

In one aspect, the approach described in WO 2011/143545 is used tosupport heterodimerization of the first heavy chain and the second heavychain of the multispecific antibody. In one aspect, amino acidmodifications in the CH3 domains of both heavy chains are introduced atpositions 368 and/or 409 (numbering according to Kabat EU index).

In one aspect, the approach described in WO 2011/090762 is used tosupport heterodimerization of the first heavy chain and the second heavychain of the bispecific antibody. WO 2011/090762 relates to amino acidmodifications according to the “knob-into-hole” (KiH) technology. In oneembodiment in the CH3 domain of one heavy chain the amino acid T atposition 366 is substituted by W, and in the CH3 domain of the otherheavy chain the amino acid Y at position 407 is substituted by A(numbering according to Kabat EU index). In another embodiment in theCH3 domain of one heavy chain the amino acid T at position 366 issubstituted by Y, and in the CH3 domain of the other heavy chain theamino acid Y at position 407 is substituted by T (numbering according toKabat EU index).

In one aspect, the approach described in WO 2009/089004 is used tosupport heterodimerization of the first heavy chain and the second heavychain of the bispecific antibody. In one embodiment in the CH3 domain ofone heavy chain the amino acid K or N at position 392 is substituted bya negatively charged amino acid (in one embodiment by E or D, in onepreferred embodiment by D), and in the CH3 domain of the other heavychain the amino acid D at position 399 the amino acid E or D at position356 or the amino acid E at position 357 is substituted by a positivelycharged amino acid (in one embodiment K or R, in one preferredembodiment by K, in one preferred embodiment the amino acids atpositions 399 or 356 are substituted by K) (numbering according to KabatEU index). In one further embodiment, in addition to the aforementionedsubstitutions, in the CH3 domain of the one heavy chain the amino acid Kor R at position 409 is substituted by a negatively charged amino acid(in one embodiment by E or D, in one preferred embodiment by D)(numbering according to Kabat EU index). In one even further aspect, inaddition to or alternatively to the aforementioned substitutions, in theCH3 domain of the one heavy chain the amino acid K at position 439and/or the amino acid K at position 370 is substituted independentlyfrom each other by a negatively charged amino acid (in one embodiment byE or D, in one preferred embodiment by D) (numbering according to KabatEU index).

In one aspect, the approach described in WO 2007/147901 is used tosupport heterodimerization of the first heavy chain and the second heavychain of the multispecific antibody. In one embodiment in the CH3 domainof one heavy chain the amino acid K at position 253 is substituted by E,the amino acid D at position 282 is substituted by K and the amino acidK at position 322 is substituted by D, and in the CH3 domain of theother heavy chain the amino acid D at position 239 is substituted by K,the amino acid E at position 240 is substituted by K and the amino acidK at position 292 is substituted by D (numbering according to Kabat EUindex).

The C-terminus of the heavy chain of the bispecific antibody as reportedherein can be a complete C-terminus ending with the amino acid residuesPGK. The C-terminus of the heavy chain can be a shortened C-terminus inwhich one or two of the C terminal amino acid residues have beenremoved. In one preferred aspect, the C-terminus of the heavy chain is ashortened C-terminus ending PG.

In one aspect of all aspects as reported herein, a bispecific antibodycomprising a heavy chain including a C-terminal CH3 domain as specifiedherein, comprises the C-terminal glycine-lysine dipeptide (G446 andK447, numbering according to Kabat EU index). In one embodiment of allaspects as reported herein, a bispecific antibody comprising a heavychain including a C-terminal CH3 domain, as specified herein, comprisesa C-terminal glycine residue (G446, numbering according to Kabat EUindex).

Modifications in the Fab Domains

In one aspect, provided is an anti-PD1/anti-LAG3 bispecific antibody,wherein in one of the Fab fragments either the variable domains VH andVL or the constant domains CH1 and CL are exchanged. The bispecificantibodies are prepared according to the Crossmab technology.

Multispecific antibodies with a domain replacement/exchange in onebinding arm (CrossMabVH-VL or CrossMabCH-CL) are described in detail inWO2009/080252, WO2009/080253 and Schaefer, W. et al, PNAS, 108 (2011)11187-1191. They clearly reduce the byproducts caused by the mismatch ofa light chain against a first antigen with the wrong heavy chain againstthe second antigen (compared to approaches without such domainexchange).

In a particular aspect, provided is an anti-PD1/anti-LAG3 bispecificantibody, wherein in one of the Fab fragments the variable domains VLand VH are replaced by each other so that the VH domain is part of thelight chain and the VL domain is part of the heavy chain. In aparticular aspect, the bispecific antibody is one, wherein in the firstFab fragment comprising the antigen binding domain that specificallybinds to PD1 the variable domains VL and VH are replaced by each other.

In another aspect, and to further improve correct pairing, theanti-PD1/anti-LAG3 bispecific antibody can contain different chargedamino acid substitutions (so-called “charged residues”). Thesemodifications are introduced in the crossed or non-crossed CH1 and CLdomains. Such modifications are described e.g. in WO2015/150447,WO2016/020309 and PCT/EP2016/073408.

In a particular aspect, provided is an anti-PD1/anti-LAG3 bispecificantibody, wherein in one of the Fab fragments in the constant domain CLthe amino acid at position 124 is substituted independently by lysine(K), arginine (R) or histidine (H) (numbering according to Kabat EUIndex), and in the constant domain CH1 the amino acids at positions 147and 213 are substituted independently by glutamic acid (E) or asparticacid (D) (numbering according to Kabat EU index). In a particularaspect, the bispecific antibody is one, wherein in the second Fabfragment comprising the antigen binding domain that specifically bindsto TIM3 the constant domain CL the amino acid at position 124 issubstituted independently by lysine (K), arginine (R) or histidine (H)(numbering according to Kabat EU Index), and in the constant domain CH1the amino acids at positions 147 and 213 are substituted independentlyby glutamic acid (E) or aspartic acid (D) (numbering according to KabatEU index).

In a particular aspect, provided is an anti-PD1/anti-LAG3 bispecificantibody, wherein in one of CL domains the amino acid at position 123(EU numbering) has been replaced by arginine (R) and the amino acid atposition 124 (EU numbering) has been substituted by lysine (K) andwherein in one of the CH1 domains the amino acids at position 147 (EUnumbering) and at position 213 (EU numbering) have been substituted byglutamic acid (E). In a particular aspect, the bispecific antibody isone, wherein in the Fab fragment comprising the antigen binding domainthat specifically binds to LAG3 the amino acid at position 123 (EUnumbering) has been replaced by arginine (R) and the amino acid atposition 124 (EU numbering) has been substituted by lysine (K) andwherein in one of the CH1 domains the amino acids at position 147 (EUnumbering) and at position 213 (EU numbering) have been substituted byglutamic acid (E).

In a further aspect, the bispecific antibody is a bivalent antibodycomprising

-   -   a) a first light chain and a first heavy chain of an antibody        specifically binding to a first antigen, and    -   b) a second light chain and a second heavy chain of an antibody        specifically binding to a second antigen, wherein the variable        domains VL and VH of the second light chain and the second heavy        chain are replaced by each other.

The antibody under a) does not contain a modification as reported underb) and the heavy chain and the light chain under a) are isolated chains.

In the antibody under b) within the light chain the variable light chaindomain VL is replaced by the variable heavy chain domain VH of saidantibody, and within the heavy chain the variable heavy chain domain VHis replaced by the variable light chain domain VL of said antibody.

In one aspect, (i) in the constant domain CL of the first light chainunder a) the amino acid at position 124 (numbering according to Kabat)is substituted by a positively charged amino acid, and wherein in theconstant domain CH1 of the first heavy chain under a) the amino acid atposition 147 or the amino acid at position 213 (numbering according toKabat EU index) is substituted by a negatively charged amino acid, or(ii) in the constant domain CL of the second light chain under b) theamino acid at position 124 (numbering according to Kabat) is substitutedby a positively charged amino acid, and wherein in the constant domainCH1 of the second heavy chain under b) the amino acid at position 147 orthe amino acid at position 213 (numbering according to Kabat EU index)is substituted by a negatively charged amino acid.

In another aspect, (i) in the constant domain CL of the first lightchain under a) the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat) (in one preferred embodiment independently by lysine(K) or arginine (R)), and wherein in the constant domain CH1 of thefirst heavy chain under a) the amino acid at position 147 or the aminoacid at position 213 is substituted independently by glutamic acid (E)or aspartic acid (D) (numbering according to Kabat EU index), or (ii) inthe constant domain CL of the second light chain under b) the amino acidat position 124 is substituted independently by lysine (K), arginine (R)or histidine (H) (numbering according to Kabat) (in one preferredembodiment independently by lysine (K) or arginine (R)), and wherein inthe constant domain CH1 of the second heavy chain under b) the aminoacid at position 147 or the amino acid at position 213 is substitutedindependently by glutamic acid (E) or aspartic acid (D) (numberingaccording to Kabat EU index).

In one aspect, in the constant domain CL of the second heavy chain theamino acids at position 124 and 123 are substituted by K (numberingaccording to Kabat EU index).

In one aspect, in the constant domain CL of the second heavy chain theamino acid at position 123 is substituted by R and the amino acid asposition 124 is substituted by K (numbering according to Kabat EUindex).

In one aspect, in the constant domain CH1 of the second light chain theamino acids at position 147 and 213 are substituted by E (numberingaccording to EU index of Kabat).

In one aspect, in the constant domain CL of the first light chain theamino acids at position 124 and 123 are substituted by K, and in theconstant domain CH1 of the first heavy chain the amino acids at position147 and 213 are substituted by E (numbering according to Kabat EUindex).

In one aspect, in the constant domain CL of the first light chain theamino acid at position 123 is substituted by R and the amino acid atposition 124 is substituted by K, and in the constant domain CH1 of thefirst heavy chain the amino acids at position 147 and 213 are bothsubstituted by E (numbering according to Kabat EU index).

In one aspect, in the constant domain CL of the second heavy chain theamino acids at position 124 and 123 are substituted by K, and wherein inthe constant domain CH1 of the second light chain the amino acids atposition 147 and 213 are substituted by E, and in the variable domain VLof the first light chain the amino acid at position 38 is substituted byK, in the variable domain VH of the first heavy chain the amino acid atposition 39 is substituted by E, in the variable domain VL of the secondheavy chain the amino acid at position 38 is substituted by K, and inthe variable domain VH of the second light chain the amino acid atposition 39 is substituted by E (numbering according to Kabat EU index).

In one aspect, the bispecific antibody is a bivalent antibody comprising

-   -   a) a first light chain and a first heavy chain of an antibody        specifically binding to a first antigen, and    -   b) a second light chain and a second heavy chain of an antibody        specifically binding to a second antigen, wherein the variable        domains VL and VH of the second light chain and the second heavy        chain are replaced by each other, and wherein the constant        domains CL and CH1 of the second light chain and the second        heavy chain are replaced by each other.

The antibody under a) does not contain a modification as reported underb) and the heavy chain and the light chain und a) are isolated chains.In the antibody under b) within the light chain the variable light chaindomain VL is replaced by the variable heavy chain domain VH of saidantibody, and the constant light chain domain CL is replaced by theconstant heavy chain domain CH1 of said antibody; and within the heavychain the variable heavy chain domain VH is replaced by the variablelight chain domain VL of said antibody, and the constant heavy chaindomain CH1 is replaced by the constant light chain domain CL of saidantibody.

In one aspect, the bispecific antibody is a bivalent antibody comprising

-   -   a) a first light chain and a first heavy chain of an antibody        specifically binding to a first antigen, and    -   b) a second light chain and a second heavy chain of an antibody        specifically binding to a second antigen, wherein the constant        domains CL and CH1 of the second light chain and the second        heavy chain are replaced by each other.

The antibody under a) does not contain a modification as reported underb) and the heavy chain and the light chain under a) are isolated chains.In the antibody under b) within the light chain the constant light chaindomain CL is replaced by the constant heavy chain domain CH1 of saidantibody; and within the heavy chain the constant heavy chain domain CH1is replaced by the constant light chain domain CL of said antibody.

In one aspect, the bispecific antibody is a bispecific antibodycomprising

-   -   a) a full length antibody specifically binding to a first        antigen and consisting of two antibody heavy chains and two        antibody light chains, and    -   b) one, two, three or four single chain Fab fragments        specifically binding to a second antigen,

wherein said single chain Fab fragments under b) are fused to said fulllength antibody under a) via a peptide linker at the C- or N-terminus ofthe heavy or light chain of said full length antibody.

In one aspect, one or two identical single chain Fab fragments bindingto a second antigen are fused to the full length antibody via a peptidelinker at the C terminus of the heavy or light chains of said fulllength antibody.

In one aspect, one or two identical single chain Fab (scFab) fragmentsbinding to a second antigen are fused to the full length antibody via apeptide linker at the C terminus of the heavy chains of said full lengthantibody.

In one aspect, one or two identical single chain Fab (scFab) fragmentsbinding to a second antigen are fused to the full length antibody via apeptide linker at the C terminus of the light chains of said full lengthantibody.

In one aspect, two identical single chain Fab (scFab) fragments bindingto a second antigen are fused to the full length antibody via a peptidelinker at the C-terminus of each heavy or light chain of said fulllength antibody

In one aspect, two identical single chain Fab (scFab) fragments bindingto a second antigen are fused to the full length antibody via a peptidelinker at the C-terminus of each heavy chain of said full lengthantibody.

In one aspect, two identical single chain Fab (scFab) fragments bindingto a second antigen are fused to the full length antibody via a peptidelinker at the C-terminus of each light chain of said full lengthantibody.

In one aspect, the bispecific antibody is a trivalent antibodycomprising

-   -   a) a full length antibody specifically binding to a first        antigen and consisting of two antibody heavy chains and two        antibody light chains,    -   b) a first polypeptide consisting of        -   ba) an antibody heavy chain variable domain (VH), or        -   bb) an antibody heavy chain variable domain (VH) and an            antibody constant domain 1 (CH1),

wherein said first polypeptide is fused with the N-terminus of its VHdomain via a peptidic linker to the C-terminus of one of the two heavychains of said full length antibody,

-   -   c) a second polypeptide consisting of        -   ca) an antibody light chain variable domain (VL), or        -   cb) an antibody light chain variable domain (VL) and an            antibody light chain constant domain (CL),

wherein said second polypeptide is fused with the N-terminus of the VLdomain via a peptide linker to the C-terminus of the other of the twoheavy chains of said full length antibody, and

wherein the antibody heavy chain variable domain (VH) of the firstpolypeptide and the antibody light chain variable domain (VL) of thesecond polypeptide together form an antigen binding domain specificallybinding to a second antigen.

In one aspect, the antibody heavy chain variable domain (VH) of thepolypeptide under b) and the antibody light chain variable domain (VL)of the polypeptide under c) are linked and stabilized via an interchaindisulfide bridge by introduction of a disulfide bond between thefollowing positions:

-   -   (i) heavy chain variable domain position 44 to light chain        variable domain position 100, or    -   (ii) heavy chain variable domain position 105 to light chain        variable domain position 43, or    -   (iii) heavy chain variable domain position 101 to light chain        variable domain position 100 (numbering always according to        Kabat EU index).

Techniques to introduce unnatural disulfide bridges for stabilizationare described e.g. in WO 94/029350, Rajagopal, V., et al., Prot. Eng.(1997) 1453-1459; Kobayashi, H., et al., Nucl. Med. Biol. 25 (1998)387-393; and Schmidt, M., et al., Oncogene 18 (1999) 1711-1721. In oneembodiment the optional disulfide bond between the variable domains ofthe polypeptides under b) and c) is between heavy chain variable domainposition 44 and light chain variable domain position 100. In oneembodiment the optional disulfide bond between the variable domains ofthe polypeptides under b) and c) is between heavy chain variable domainposition 105 and light chain variable domain position 43 (numberingalways according to Kabat). In one embodiment a trivalent, bispecificantibody without said optional disulfide stabilization between thevariable domains VH and VL of the single chain Fab fragments ispreferred.

In one aspect, the bispecific antibody is a trispecific or tetraspecificantibody, comprising

-   -   a) a first light chain and a first heavy chain of a full length        antibody which specifically binds to a first antigen, and    -   b) a second (modified) light chain and a second (modified) heavy        chain of a full length antibody which specifically binds to a        second antigen, wherein the variable domains VL and VH are        replaced by each other, and/or wherein the constant domains CL        and CH1 are replaced by each other, and    -   c) wherein one to four antigen binding domains which        specifically bind to one or two further antigens (i.e. to a        third and/or fourth antigen) are fused via a peptide linker to        the C- or N-terminus of the light chains or heavy chains of a)        and/or b).

The antibody under a) does not contain a modification as reported underb) and the heavy chain and the light chain und a) are isolated chains.

In one aspect, the trispecific or tetraspecific antibody comprises underc) one or two antigen binding domains which specifically bind to one ortwo further antigens.

In one aspect, the antigen binding domains are selected from the groupof a scFv fragment and a scFab fragment.

In one aspect, the antigen binding domains are scFv fragments.

In one aspect, the antigen binding domains are scFab fragments.

In one aspect, the antigen binding domains are fused to the C-terminusof the heavy chains of a) and/or b).

In one aspect, the trispecific or tetraspecific antibody comprises underc) one or two antigen binding domains which specifically bind to onefurther antigen.

In one aspect, the trispecific or tetraspecific antibody comprises underc) two identical antigen binding domains which specifically bind to athird antigen. In one preferred embodiment such two identical antigenbinding domains are fused both via the same peptidic linker to theC-terminus of the heavy chains of a) and b). In one preferred embodimentthe two identical antigen binding domains are either a scFv fragment ora scFab fragment.

In one aspect, the trispecific or tetraspecific antibody comprises underc) two antigen binding domains which specifically bind to a third and afourth antigen. In one embodiment said two antigen binding domains arefused both via the same peptide connector to the C-terminus of the heavychains of a) and b). In one preferred embodiment said two antigenbinding domains are either a scFv fragment or a scFab fragment.

In one aspect, the bispecific antibody is a bispecific, tetravalentantibody comprising

-   -   a) two light chains and two heavy chains of an antibody, which        specifically bind to a first antigen (and comprise two Fab        fragments),    -   b) two additional Fab fragments of an antibody, which        specifically bind to a second antigen, wherein said additional        Fab fragments are fused both via a peptidic linker either to the        C- or N-termini of the heavy chains of a), and

wherein in the Fab fragments the following modifications were performed

-   -   (i) in both Fab fragments of a), or in both Fab fragments of b),        the variable domains VL and VH are replaced by each other,        and/or the constant domains CL and CH1 are replaced by each        other, or    -   (ii) in both Fab fragments of a) the variable domains VL and VH        are replaced by each other, and the constant domains CL and CH1        are replaced by each other, and in both Fab fragments of b) the        variable domains VL and VH are replaced by each other, or the        constant domains CL and CH1 are replaced by each other, or    -   (iii) in both Fab fragments of a) the variable domains VL and VH        are replaced by each other, or the constant domains CL and CH1        are replaced by each other, and in both Fab fragments of b) the        variable domains VL and VH are replaced by each other, and the        constant domains CL and CH1 are replaced by each other, or    -   (iv) in both Fab fragments of a) the variable domains VL and VH        are replaced by each other, and in both Fab fragments of b) the        constant domains CL and CH1 are replaced by each other, or    -   (v) in both Fab fragments of a) the constant domains CL and CH1        are replaced by each other, and in both Fab fragments of b) the        variable domains VL and VH are replaced by each other.

In one aspect, said additional Fab fragments are fused both via apeptidic linker either to the C-termini of the heavy chains of a), or tothe N-termini of the heavy chains of a).

In one aspect, said additional Fab fragments are fused both via apeptidic linker either to the C-termini of the heavy chains of a).

In one aspect, said additional Fab fragments are fused both via apeptide linker to the N-termini of the heavy chains of a).

In one aspect, in the Fab fragments the following modifications areperformed: in both Fab fragments of a), or in both Fab fragments of b),the variable domains VL and VH are replaced by each other, and/or theconstant domains CL and CH1 are replaced by each other.

In one aspect, the bispecific antibody is a tetravalent antibodycomprising:

-   -   a) a (modified) heavy chain of a first antibody, which        specifically binds to a first antigen and comprises a first        VH-CHT domain pair, wherein to the C terminus of said heavy        chain the N-terminus of a second VH-CHT domain pair of said        first antibody is fused via a peptide linker,    -   b) two light chains of said first antibody of a),    -   c) a (modified) heavy chain of a second antibody, which        specifically binds to a second antigen and comprises a first        VH-CL domain pair, wherein to the C-terminus of said heavy chain        the N-terminus of a second VH-CL domain pair of said second        antibody is fused via a peptide linker, and    -   d) two (modified) light chains of said second antibody of c),        each comprising a CL-CHT domain pair.

In one aspect, the bispecific antibody comprises

-   -   a) the heavy chain and the light chain of a first full length        antibody that specifically binds to a first antigen, and    -   b) the heavy chain and the light chain of a second full length        antibody that specifically binds to a second antigen, wherein        the N-terminus of the heavy chain is connected to the C-terminus        of the light chain via a peptide linker.

The antibody under a) does not contain a modification as reported underb) and the heavy chain and the light chain are isolated chains.

In one aspect, the bispecific antibody comprises

-   -   a) a full length antibody specifically binding to a first        antigen and consisting of two antibody heavy chains and two        antibody light chains, and    -   b) an Fv fragment specifically binding to a second antigen        comprising a VH2 domain and a VL2 domain, wherein both domains        are connected to each other via a disulfide bridge,    -   wherein only either the VH2 domain or the VL2 domain is fused        via a peptide linker to the heavy or light chain of the full        length antibody specifically binding to a first antigen.

In the bispecific antibody the heavy chains and the light chains undera) are isolated chains.

In one aspect, the other of the VH2 domain or the VL2 domain is notfused via a peptide linker to the heavy or light chain of the fulllength antibody specifically binding to a first antigen.

In all aspects as reported herein the first light chain comprises a VLdomain and a CL domain and the first heavy chain comprises a VH domain,a CH1 domain, a hinge region, a CH2 domain and a CH3 domain.

In one aspect, the bispecific antibody is a trivalent antibodycomprising

-   -   a) two Fab fragments that specifically binds to a first antigen,    -   b) one CrossFab fragment that specifically binds to a second        antigen in which the CH1 and the CL domain are exchanged for        each other,    -   c) one Fc-region comprising a first Fc-region heavy chain and a        second Fc region heavy chain,

wherein the C-terminus of CH1 domains of the two Fab fragments areconnected to the N-terminus of the heavy chain Fc-region polypeptides,and wherein the C-terminus of the CL domain of the CrossFab fragment isconnected to the N-terminus of the VH domain of one of the Fabfragments.

In one aspect, the bispecific antibody is a trivalent antibodycomprising

-   -   a) two Fab fragments that specifically binds to a first antigen,    -   b) one CrossFab fragment that specifically binds to a second        antigen in which the CH1 and the CL domain are exchanged for        each other,    -   c) one Fc-region comprising a first Fc-region heavy chain and a        second Fc region heavy chain,

wherein the C-terminus of CH1 domain of the first Fab fragment isconnected to the N-terminus of one of the heavy chain Fc-regionpolypeptides and the C-terminus of the CL-domain of the CrossFabfragment is connected to the N-terminus of the other heavy chainFc-region polypeptide, and wherein the C-terminus of the CH1 domain ofthe second Fab fragment is connected to the N-terminus of the VH domainof the first Fab fragment or to the N-terminus of the VH domain of theCrossFab fragment.

In one aspect, the bispecific antibody comprises

-   -   a) a full length antibody specifically binding to a first        antigen and consisting of two antibody heavy chains and two        antibody light chains, and    -   b) a Fab fragment specifically binding to a second antigen        comprising a VH2 domain and a VL2 domain comprising a heavy        chain fragment and a light chain fragment, wherein within the        light chain fragment the variable light chain domain VL2 is        replaced by the variable heavy chain domain VH2 of said        antibody, and within the heavy chain fragment the variable heavy        chain domain VH2 is replaced by the variable light chain domain        VL2 of said antibody

wherein the heavy chain Fab fragment is inserted between the CH1 domainof one of the heavy chains of the full length antibody and therespective Fc-region of the full length antibody, and the N-terminus ofthe light chain Fab fragment is conjugated to the C-terminus of thelight chain of the full length antibody that is paired with the heavychain of the full length antibody into which the heavy chain Fabfragment has been inserted.

In one aspect, the bispecific antibody comprises

-   -   a) a full length antibody specifically binding to a first        antigen and consisting of two antibody heavy chains and two        antibody light chains, and    -   b) a Fab fragment specifically binding to a second antigen        comprising a VH2 domain and a VL2 domain comprising a heavy        chain fragment and a light chain fragment, wherein within the        light chain fragment the variable light chain domain VL2 is        replaced by the variable heavy chain domain VH2 of said        antibody, and within the heavy chain fragment the variable heavy        chain domain VH2 is replaced by the variable light chain domain        VL2 of said antibody and wherein the C-terminus of the heavy        chain fragment of the Fab fragment is conjugated to the        N-terminus of one of the heavy chains of the full length        antibody and the C-terminus of the light chain fragment of the        Fab fragment is conjugated to the N-terminus of the light chain        of the full length antibody that pairs with the heavy chain of        the full length antibody to which the heavy chain fragment of        the Fab fragment is conjugated.

Polynucleotides

Provided are furthermore isolated polynucleotides encoding a bispecificantibody as described herein or a fragment thereof.

The term “nucleic acid molecule” or “polynucleotide” includes anycompound and/or substance that comprises a polymer of nucleotides. Eachnucleotide is composed of a base, specifically a purine- or pyrimidinebase (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil(U)), a sugar (i.e. deoxyribose or ribose), and a phosphate group.Often, the nucleic acid molecule is described by the sequence of bases,whereby said bases represent the primary structure (linear structure) ofa nucleic acid molecule. The sequence of bases is typically representedfrom 5′ to 3′. Herein, the term nucleic acid molecule encompassesdeoxyribonucleic acid (DNA) including e.g., complementary DNA (cDNA) andgenomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA),synthetic forms of DNA or RNA, and mixed polymers comprising two or moreof these molecules. The nucleic acid molecule may be linear or circular.In addition, the term nucleic acid molecule includes both, sense andantisense strands, as well as single stranded and double stranded forms.Moreover, the herein described nucleic acid molecule can containnaturally occurring or non-naturally occurring nucleotides. Examples ofnon-naturally occurring nucleotides include modified nucleotide baseswith derivatized sugars or phosphate backbone linkages or chemicallymodified residues. Nucleic acid molecules also encompass DNA and RNAmolecules which are suitable as a vector for direct expression of anantibody of the invention in vitro and/or in vivo, e.g., in a host orpatient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors, can beunmodified or modified. For example, mRNA can be chemically modified toenhance the stability of the RNA vector and/or expression of the encodedmolecule so that mRNA can be injected into a subject to generate theantibody in vivo (see e.g., Stadler ert al, Nature Medicine 2017,published online 12 Jun. 2017, doi:10.1038/nm.4356 or EP 2 101 823 B1).

An “isolated” polynucleotide refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatedpolynucleotide includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

The isolated polynucleotides encoding bispecific antibodies of theinvention may be expressed as a single polynucleotide that encodes theentire antigen binding molecule or as multiple (e.g., two or more)polynucleotides that are co-expressed. Polypeptides encoded bypolynucleotides that are co-expressed may associate through, e.g.,disulfide bonds or other means to form a functional antigen bindingmolecule. For example, the light chain portion of an immunoglobulin maybe encoded by a separate polynucleotide from the heavy chain portion ofthe immunoglobulin. When co-expressed, the heavy chain polypeptides willassociate with the light chain polypeptides to form the immunoglobulin.

In some aspects, the isolated polynucleotide encodes a polypeptidecomprised in the bispecific antibody according to the invention asdescribed herein.

In one aspect, the isolated polynucleotides are provided encoding ananti-PD1/anti-LAG3 bispecific antibody, wherein said first antigenbinding domain specifically binding to PD1 comprises a VH domaincomprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:1,(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (iii)HVR-H3 comprising an amino acid sequence of SEQ ID NO:3; and a VL domaincomprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:4;(ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (iii)HVR-L3 comprising the amino acid sequence of SEQ ID NO:6.

Preparation of Bispecific Antibodies for Use in the Invention

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. For these methods one ormore isolated nucleic acid(s) encoding an antibody are provided.

In case of a native antibody or native antibody fragment two nucleicacids are required, one for the light chain or a fragment thereof andone for the heavy chain or a fragment thereof. Such nucleic acid(s)encode an amino acid sequence comprising the VL and/or an amino acidsequence comprising the VH of the antibody (e.g., the light and/or heavychain(s) of the antibody). These nucleic acids can be on the sameexpression vector or on different expression vectors. In case of certainbispecific antibodies with heterodimeric heavy chains four nucleic acidsare required, one for the first light chain, one for the first heavychain comprising the first hetreomonomeric Fc-region polypeptide, onefor the second light chain, and one for the second heavy chaincomprising the second heteromonomeric Fc-region polypeptide. The fournucleic acids can be comprised in one or more nucleic acid molecules orexpression vectors. For example, such nucleic acid(s) encode an aminoacid sequence comprising the first VL and/or an amino acid sequencecomprising the first VH including the first heteromonomeric Fc-regionand/or an amino acid sequence comprising the second VL and/or an aminoacid sequence comprising the second VH including the secondheteromonomeric Fc-region of the antibody (e.g., the first and/or secondlight and/or the first and/or second heavy chains of the antibody).These nucleic acids can be on the same expression vector or on differentexpression vectors, normally these nucleic acids are located on two orthree expression vectors, i.e. one vector can comprise more than one ofthese nucleic acids. Examples of these bispecific antibodies areCrossMabs and T-cell bispecifics (see, e.g. Schaefer, W. et al, PNAS,108 (2011) 11187-1191). For example, one of the heteromonomeric heavychain comprises the so-called “knob mutations” (T366W and optionally oneof S354C or Y349C) and the other comprises the so-called “holemutations” (T366S, L368A and Y407V and optionally Y349C or S354C) (see,e.g., Carter, P. et al., Immunotechnol. 2 (1996) 73).

In one aspect, isolated nucleic acid encoding a bispecific antibodydescribed herein is provided. Such nucleic acid may encode an amino acidsequence comprising the VL and/or an amino acid sequence comprising theVH of the antigen binding domains that specifically bind to PD1 andLAG3, respectively (e.g., in the light and/or heavy chains of theantibody). In a further aspect, one or more vectors (e.g., expressionvectors) comprising such nucleic acid are provided. In a further aspect,a host cell comprising such nucleic acid is provided. In one suchaspect, a host cell comprises (e.g., has been transformed with): (1) afirst vector comprising a first pair of nucleic acids that encode aminoacid sequences one of them comprising the first VL and the othercomprising the first VH of the antibody and a second vector comprising asecond pair of nucleic acids that encode amino acid sequences one ofthem comprising the second VL and the other comprising the second VH ofthe antibody, or (2) a first vector comprising a first nucleic acid thatencode an amino acid sequence comprising one of the variable domains(preferably a light chain variable domain), a second vector comprising apair of nucleic acids that encode amino acid sequences one of themcomprising a light chain variable domain and the other comprising thefirst heavy chain variable domain, and a third vector comprising a pairof nucleic acids that encode amino acid sequences one of them comprisingthe respective other light chain variable domain as in the second vectorand the other comprising the second heavy chain variable domain, or (3)a first vector comprising a nucleic acid that encodes an amino acidsequence comprising the first VL of the antibody, a second vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe first VH of the antibody, a third vector comprising a nucleic acidthat encodes an amino acid sequence comprising the second VL of theantibody, and a fourth vector comprising a nucleic acid that encodes anamino acid sequence comprising the second VH of the antibody. In oneaspect, the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO)cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one aspect, amethod of making a bispecific antibody is provided, wherein the methodcomprises culturing a host cell comprising a nucleic acid encoding theantibody, as provided above, under conditions suitable for expression ofthe antibody, and optionally recovering the antibody from the host cell(or host cell culture medium).

For recombinant production of the anti-CD20/anti-CD3 bispecificantibodies or anti-PD1/anti-LAG3 bispecific antibodies described herein,nucleic acid encoding the bispecific antibodies, e.g., as describedabove, is isolated and inserted into one or more vectors for furthercloning and/or expression in a host cell. Such nucleic acid may bereadily isolated and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically togenes encoding the heavy and light chains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, K. A., In:Methods in Molecular Biology, Vol. 248, Lo, B. K. C. (ed.), HumanaPress, Totowa, NJ (2003), pp. 245-254, describing expression of antibodyfragments in E. coli.) After expression, the antibody may be isolatedfrom the bacterial cell paste in a soluble fraction and can be furtherpurified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gemgross, T. U., Nat. Biotech. 22 (2004) 1409-1414; and Li,H. et al., Nat. Biotech. 24 (2006) 210-215.

Suitable host cells for the expression of glycosylated antibodies arealso derived from multicellular organisms (invertebrates andvertebrates). Examples of invertebrate cells include plant and insectcells. Numerous baculoviral strains have been identified which may beused in conjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham, F. L. et al., J. Gen Virol. 36(1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4cells as described, e.g., in Mather, J. P., Biol. Reprod. 23 (1980)243-252); monkey kidney cells (CV1); African green monkey kidney cells(VERO-76); human cervical carcinoma cells (HELA); canine kidney cells(MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); humanliver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, asdescribed, e.g., in Mather, J. P. et al., Annals N.Y. Acad. Sci. 383(1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian hostcell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHOcells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980)4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2/0. For areview of certain mammalian host cell lines suitable for antibodyproduction, see, e.g., Yazaki, P. and Wu, A. M., Methods in MolecularBiology, Vol. 248, Lo, B. K. C. (ed.), Humana Press, Totowa, NJ (2004),pp. 255-268.

Assays

The bispecific antibodies provided herein may be identified, screenedfor, or characterized for their physical/chemical properties and/orbiological activities by various assays known in the art.

1. Affinity Assays

The affinity of the bispecific antigen binding molecules, antibodies andantibody fragments provided herein for the corresponding antigens can bedetermined in accordance with the methods set forth in the Examples bysurface plasmon resonance (SPR), using standard instrumentation such asa Biacore® instrument (GE Healthcare), and receptors or target proteinssuch as may be obtained by recombinant expression. A specificillustrative and exemplary embodiment for measuring binding affinity hasbeen described in Examples 2, 8 or 11 of WO WO 2018/185043. According toone aspect, K_(D) is measured by surface plasmon resonance using aBIACORE® T100 machine (GE Healthcare) at 25° C.

2. Binding Assays and Other Assays

In one aspect, the bispecific antibodies of the invention are tested forits antigen binding activity, e.g., by known methods such as ELISA,Western blot, etc. The binding of the anti-PD1/anti-LAG3 bispecificantibodies provided herein to the corresponding recombinant antigen orto antigen-expressing cells may be evaluated by ELISA as described inExamples 8 or 11 of WO 2018/185043. In a further aspect, freshperipheral blood mononuclear cells (PBMCs) can be used in binding assaysto show binding to different peripheral blood mononuclear cells (PBMC)such as monocytes, NK cells and T cells.

In another aspect, a cellular dimerization assay was used to demonstratethe dimerization or at last binding/interaction of two differentreceptors PD1 and LAG3, which are cytosolically fused with two fragmentsof an enzyme, upon ligation or cross-linking with a bispecific antibodyagainst both targets. Hereby only one receptor alone shows no enzymaticactivity. For this specific interaction, the cytosolic C-terminal endsof both receptors were individually fused to heterologous subunits of areporter enzyme. A single enzyme subunit alone showed no reporteractivity. However, simultaneous binding to both receptors was expectedto lead to local cytocolic accumulation of both receptors,complementation of the two heterologous enzyme subunits, and finally toresult in the formation of a specific and functional enzyme thathydrolyzes a substrate thereby generating a chemiluminescent signal(Example 11 of WO 2018/185043).

3. Activity Assays

In one aspect, assays are provided for identifying an anti-PD1/anti-LAG3bispecific antibody having biological activity. Biological activity mayinclude, e.g., the ability to enhance the activation and/orproliferation of different immune cells, especially T-cells, secretionof immune-modulating cytokines such IFNγ or TNF-alpha, blocking the PD1pathway, blocking the LAG3 pathway, killing of tumor cells. Antibodieshaving such biological activity in vivo and/or in vitro are alsoprovided. In certain aspects, an antibody of the invention is tested forsuch biological activity. In one aspect, provided is an immune cellassay which measures the activation of lymphocytes from one individual(donor X) to lymphocytes from another individual (donor Y). The mixedlymphocyte reaction (MLR) can demonstrate the effect of blocking the PD1pathway to lymphocyte effector cells. T cells in the assay were testedfor activation and their IFN-gamma secretion in the presence or absenceof bispecific antibodies of the invention. The assay is described inmore detail in Example 9 of WO 2018/185043.

Pharmaceutical Compositions, Formulations and Routes of Administration

In a further aspect, the invention provides pharmaceutical compositionscomprising the anti-CD20/anti-CD3 antibodies and anti-PD1/anti-LAG3antibodies provided herein, e.g., for use in any of the belowtherapeutic methods. In one embodiment, a pharmaceutical compositioncomprises an anti-CD20/anti-CD3 antibody and an anti-PD1/anti-LAG3antibody provided herein and at least one pharmaceutically acceptableexcipient. In another embodiment, a pharmaceutical composition comprisesan antibody provided herein and at least one additional therapeuticagent, e.g., as described below.

Pharmaceutical compositions of the present invention comprise atherapeutically effective amount of one or more bispecific antibodiesdissolved or dispersed in a pharmaceutically acceptable excipient. Thephrases “pharmaceutical or pharmacologically acceptable” refers tomolecular entities and compositions that are generally non-toxic torecipients at the dosages and concentrations employed, i.e. do notproduce an adverse, allergic or other untoward reaction whenadministered to an animal, such as, for example, a human, asappropriate. The preparation of a pharmaceutical composition thatcontains at least one antibody and optionally an additional activeingredient will be known to those of skill in the art in light of thepresent disclosure, as exemplified by Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990, incorporated herein byreference. In particular, the compositions are lyophilized formulationsor aqueous solutions. As used herein, “pharmaceutically acceptableexcipient” includes any and all solvents, buffers, dispersion media,coatings, surfactants, antioxidants, preservatives (e.g. antibacterialagents, antifungal agents), isotonic agents, salts, stabilizers andcombinations thereof, as would be known to one of ordinary skill in theart.

Parenteral compositions include those designed for administration byinjection, e.g. subcutaneous, intradermal, intralesional, intravenous,intraarterial intramuscular, intrathecal or intraperitoneal injection.For injection, the antigen binding molecules of the invention may beformulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiological saline buffer. The solution may contain formulatory agentssuch as suspending, stabilizing and/or dispersing agents. Alternatively,the fusion proteins may be in powder form for constitution with asuitable vehicle, e.g., sterile pyrogen-free water, before use. Sterileinjectable solutions are prepared by incorporating the fusion proteinsof the invention in the required amount in the appropriate solvent withvarious of the other ingredients enumerated below, as required.Sterility may be readily accomplished, e.g., by filtration throughsterile filtration membranes. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The composition must be stable under theconditions of manufacture and storage, and preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Itwill be appreciated that endotoxin contamination should be keptminimally at a safe level, for example, less that 0.5 ng/mg protein.Suitable pharmaceutically acceptable excipients include, but are notlimited to: buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Aqueous injectionsuspensions may contain compounds which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, dextran,or the like. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl cleats or triglycerides, or liposomes.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(18th Ed. Mack Printing Company, 1990). Sustained-release preparationsmay be prepared. Suitable examples of sustained-release preparationsinclude semipermeable matrices of solid hydrophobic polymers containingthe polypeptide, which matrices are in the form of shaped articles, e.g.films, or microcapsules. In particular embodiments, prolonged absorptionof an injectable composition can be brought about by the use in thecompositions of agents delaying absorption, such as, for example,aluminum monostearate, gelatin or combinations thereof.

Exemplary pharmaceutically acceptable excipients herein further includeinsterstitial drug dispersion agents such as soluble neutral-activehyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, BaxterInternational, Inc.). Certain exemplary sHASEGPs and methods of use,including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulationsincluding a histidine-acetate buffer.

In addition to the compositions described previously, the bispecificantibodies may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the fusion proteins may be formulated with suitablepolymeric or hydrophobic materials (for example as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

Pharmaceutical compositions comprising the bispecific antigen bindingmolecules of the invention may be manufactured by means of conventionalmixing, dissolving, emulsifying, encapsulating, entrapping orlyophilizing processes. Pharmaceutical compositions may be formulated inconventional manner using one or more physiologically acceptablecarriers, diluents, excipients or auxiliaries which facilitateprocessing of the proteins into preparations that can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen.

The bispecific antibodies disclosed herein may be formulated into acomposition in a free acid or base, neutral or salt form.Pharmaceutically acceptable salts are salts that substantially retainthe biological activity of the free acid or base. These include the acidaddition salts, e.g. those formed with the free amino groups of aproteinaceous composition, or which are formed with inorganic acids suchas for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine. Pharmaceutical salts tend to be more soluble in aqueous andother protic solvents than are the corresponding free base forms.

The composition herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such active ingredients are suitably present in combination inamounts that are effective for the purpose intended.

In one aspect, there is provided a pharmaceutical composition comprisingan anti-CD20/anti-CD3 bispecific antibody and a pharmaceuticallyacceptable carrier, and a second medicament comprising ananti-PD1/anti-LAG3 antibody as described herein. In one aspect, thepharmaceutical composition is for use in the treatment of aCD20-expressing cancer. In a particular aspect, the pharmaceuticalcomposition is for use in the treatment of B-cell proliferativedisorders, in particular a disease selected from the group consisting ofNon-Hodgkin lymphoma (NHL), acute lymphocytic leukemia (ALL), chroniclymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL),follicular lymphoma (FL), mantle-cell lymphoma (MCL), marginal zonelymphoma (MZL), Multiple myeloma (MM) and Hodgkin lymphoma (HL).

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

Administration of the Anti-CD20/Anti-CD3 Bispecific Antibody and theAnti-PD1/Anti-LAG3 Antibody

Both the anti-CD20/anti-CD3 bispecific antibody and theanti-PD1/anti-LAG3 antibody (both called substance herein) can beadministered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. The methods described herein areparticularly useful, however, in relation to therapeutic agentsadministered by parenteral, particularly intravenous, infusion.

Parenteral infusions include intramuscular, intravenous, intraarterial,intraperitoneal, or subcutaneous administration. Dosing can be by anysuitable route, e.g. by injections, such as intravenous or subcutaneousinjections, depending in part on whether the administration is brief orchronic. Various dosing schedules including but not limited to single ormultiple administrations over various time-points, bolus administration,and pulse infusion are contemplated herein. In one aspect, thetherapeutic agent is administered parenterally, particularlyintravenously. In a particular aspect, the substance is administered byintravenous infusion. In another aspect, the substance is administeredsubcutaneously.

Both the anti-CD20/anti-CD3 bispecific antibody and theanti-PD1/anti-LAG3 antibody would be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Boththe anti-CD20/anti-CD3 bispecific antibody and the anti-PD1/anti-LAG3antibody need not be, but are optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount oftherapeutic agent present in the formulation, the type of disorder ortreatment, and other factors discussed above. These are generally usedin the same dosages and with administration routes as described herein,or about from 1 to 99% of the dosages described herein, or in any dosageand by any route that is empirically/clinically determined to beappropriate.

For the prevention or treatment of disease, the appropriate dosage ofthe anti-CD20/anti-CD3 bispecific antibody and the anti-PD1/anti-LAG3antibody (when used in their combination or with one or more otheradditional therapeutic agents) will depend on the type of disease to betreated, the type of anti-CD20/anti-CD3 bispecific antibody, theseverity and course of the disease, whether both agents are administeredfor preventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the therapeutic agent, and thediscretion of the attending physician. Each substance is suitablyadministered to the patient at one time or over a series of treatments.Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of the substance can be an initialcandidate dosage for administration to the subject, whether, forexample, by one or more separate administrations, or by continuousinfusion. One typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment would generally be sustained until a desired suppressionof disease symptoms occurs. One exemplary dosage of the bispecificantibody would be in the range from about 0.005 mg/kg to about 10 mg/kg.In other examples, a dose may also comprise from about 1 μg/kg bodyweight, about 5 μg/kg body weight, about 10 μg/kg body weight, about 50μg/kg body weight, about 100 μg/kg body weight, about 200 μg/kg bodyweight, about 350 μg/kg body weight, about 500 μg/kg body weight, about1 mg/kg body weight, about 5 mg/kg body weight, about 10 mg/kg bodyweight, about 50 mg/kg body weight, about 100 mg/kg body weight, about200 mg/kg body weight, about 350 mg/kg body weight, about 500 mg/kg bodyweight, to about 1000 mg/kg body weight or more per administration, andany range derivable therein. In examples of a derivable range from thenumbers listed herein, a range of about 5 mg/kg body weight to about 100mg/kg body weight, about 5 μg/kg body weight to about 500 mg/kg bodyweight etc., can be administered, based on the numbers described above.Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 5.0 mg/kg or 10mg/kg (or any combination thereof) may be administered to the patient.Such doses may be administered intermittently, e.g. every week or everythree weeks (e.g. such that the patient receives from about two to abouttwenty, or e.g. about six doses of the antibody). An initial higherloading dose, followed by one or more lower doses may be administered.However, other dosage regimens may be useful. The progress of thistherapy is easily monitored by conventional techniques and assays.However, other dosage regimens may be useful. The progress of thistherapy is easily monitored by conventional techniques and assays.

In one aspect, the administration of both the anti-CD20/anti-CD3bispecific antibody and the anti-PD1/anti-LAG3 antibody is a singleadministration. In certain aspects, the administration of thetherapeutic agent is two or more administrations. In one such aspect,the substances are administered every week, every two weeks, or everythree weeks, particularly every two weeks. In one aspect, the substanceis administered in a therapeutically effective amount. In one aspect thesubstance is administered at a dose of about 10 μg/kg, about 100 μg/kg,about 200 μg/kg, about 300 μg/kg, about 400 μg/kg, about 500 μg/kg,about 600 μg/kg, about 700 μg/kg, about 800 μg/kg, about 900 μg/kg orabout 1000 μg/kg. In one embodiment, the anti-CD20/anti-CD3 bispecificantibody is administered at a dose which is higher than the dose of theanti-CD20/anti-CD3 bispecific antibody in a corresponding treatmentregimen without the administration of the anti-PD1/anti-LAG3 antibody.In one aspect, the administration of the anti-CD20/anti-CD3 bispecificantibody comprises an initial administration of a first dose of theanti-CD20/anti-CD3 bispecific antibody, and one or more subsequentadministrations of a second dose of the anti-CD20/anti-CD3 bispecificantibody, wherein the second dose is higher than the first dose. In oneaspect, the administration of the anti-CD20/anti-CD3 bispecific antibodycomprises an initial administration of a first dose of theanti-CD20/anti-CD3 bispecific antibody, and one or more subsequentadministrations of a second dose of the anti-CD20/anti-CD3 bispecificantibody, wherein the first dose is not lower than the second dose.

In one aspect, the administration of the anti-CD20/anti-CD3 bispecificantibody in the treatment regimen according to the invention is thefirst administration of the anti-CD20/anti-CD3 bispecific antibody tothe subject (at least within the same course of treatment). In oneaspect, no administration of the anti-PD1/anti-LAG3 antibody is made tothe subject prior to the administration of the anti-CD20/anti-CD3bispecific antibody. In another aspect, the anti-PD1/anti-LAG3 antibodyis administered prior to the administration of the anti-CD20/anti-CD3bispecific antibody.

In another aspect, the anti-CD20/anti-CD3 bispecific antibody is for usein combination with the anti-PD1/anti-LAG3 antibody, wherein apretreatment with an Type II anti-CD20 antibody, preferablyobinutuzumab, is performed prior to the combination treatment, whereinthe period of time between the pretreatment and the combinationtreatment is sufficient for the reduction of B-cells in the individualin response to the Type II anti-CD20 antibody, preferably obinutuzumab.

Activation of T cells can lead to severe cytokine release syndrome(CRS). In a phase 1 study conducted by TeGenero (Suntharalingam et al.,N Engl J Med (2006) 355,1018-1028), all 6 healthy volunteers experiencednear fatal, severe cytokine release syndrome (CRS) rapidly post-infusionof an inappropriately-dosed, T-cell stimulating super-agonist anti-CD28monoclonal antibody. The cytokine release associated with administrationof a T-cell activating therapeutic agent, such as the anti-CD20/anti-CD3bispecific antibody, to a subject can be significantly reduced bypre-treatment of said subject with a Type II anti-CD20 antibody, such asobinutuzumab. the use of GAZYVA® pre-treatment (Gpt) should aid in therapid depletion of B cells, both in the peripheral blood and insecondary lymphoid organs, such that the risk of highly relevant adverseevents (AEs) from strong systemic T cell activation by T-cell activatingtherapeutic agents (e.g. CRS) is reduced, while supporting exposurelevels of T-cell activating therapeutic agents that are high enough fromthe start of dosing to mediate tumour cell elimination. To date, thesafety profile of obinutuzumab (including cytokine release) has beenassessed and managed in hundreds of patients in ongoing obinutuzumabclinical trials. Finally, in addition to supporting the safety profileof T-cell activating therapeutic agents such as the anti-CD20/anti-CD3bispecific antibody, Gpt should also help prevent the formation ofanti-drug antibodies (ADAs) to these unique molecules.

In the present invention, the combination of the anti-CD20/anti-CD3bispecific antibody and the anti-PD1/anti-LAG3 antibody can be used incombination with one or more further agents in a therapy. For instance,at least one additional therapeutic agent may be co-administered. Incertain aspects, an additional therapeutic agent is an immunotherapeuticagent.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the therapeutic agent can occur prior to,simultaneously, and/or following, administration of an additionaltherapeutic agent or agents. In one embodiment, administration of thetherapeutic agent and administration of an additional therapeutic agentoccur within about one month, or within about one, two or three weeks,or within about one, two, three, four, five, or six days, of each other.

Therapeutic Methods and Compositions

CD20 is expressed on most B-cells (pan-B-cell marker) with the exceptionof stem cells and plasma cells, and are frequently expressed on mosthuman B-cell malignancies (tumor associated antigen), such as lymphomaand leukemias except for multiple myeloma, e.g. in non-Hodgkin lymphomaand acute lymphoblastic leukemia.

In one aspect, there is provided a method for treating or delayingprogression of CD20-expressing cancer in a subject comprisingadministering to the subject an effective amount of ananti-CD20/anti-CD3 antibody and an effective amount of ananti-PD1/anti-LAG3 antibody.

In one such aspect, the method further comprises administering to thesubject an effective amount of at least one additional therapeuticagent. In further embodiments, herein is provided a method for depletingB-cells comprising administering to the subject an effective amount ofan anti-CD20/anti-CD3 antibody and an effective amount of ananti-PD1/anti-LAG3 antibody. An “individual” or a “subject” according toany of the above aspects is preferably a human.

In further aspects, a composition for use in cancer immunotherapy isprovided comprising an anti-CD20/anti-CD3 antibody and ananti-PD1/anti-LAG3 antibody. In certain embodiments, a compositioncomprising an anti-CD20/anti-CD3 antibody and an effective amount of ananti-PD1/anti-LAG3 antibody for use in a method of cancer immunotherapyis provided.

In a further aspect, herein is provided the use of a compositioncomprising an anti-CD20/anti-CD3 antibody and an effective amount of ananti-PD1/anti-LAG3 antibody in the manufacture or preparation of amedicament. In one aspect, the medicament is for treatment of aCD20-expressing cancer. In one aspect, the medicament is for treatmentof a B-cell proliferative disorder. In a further aspect, the medicamentis for use in a method of treating a B-cell proliferative disordercomprising administering to an individual having a B-cell proliferativedisorder an effective amount of the medicament. In one such aspect, themethod further comprises administering to the individual an effectiveamount of at least one additional therapeutic agent. In a furtheraspect, the medicament is for depleting B-cells. B-cell proliferativedisorders are selected from the group consisting of Non-Hodgkin lymphoma(NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia(CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL),mantle-cell lymphoma (MCL), marginal zone lymphoma (MZL), Multiplemyeloma (MM) and Hodgkin lymphoma (HL). In one particular aspect, theB-cell cancer is non-Hodgkin lymphoma or acute lymphoblastic leukemia.

In a further aspect, herein is provided a method for treating a B-cellcancer. In one embodiment, the method comprises administering to anindividual having such B-cell cancer an effective amount of an effectiveamount of an anti-PD1/anti-LAG3 antibody. In one such embodiment, themethod further comprises administering to the individual an effectiveamount of at least one additional therapeutic agent, as described below.An “individual” according to any of the above embodiments may be ahuman. In particular, the B-cell cancer is a B-cell lymphoma or a B-cellleukemia. In one aspect, the B-cell cancer is non-Hodgkin lymphoma oracute lymphoblastic leukemia.

The combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the anti-PD1/anti-LAG3 bispecific antibody as reportedherein can occur prior to, simultaneously, and/or following,administration of the additional therapeutic agent or agents. In oneaspect, administration of the effective amount of an anti-CD20/anti-CD3bispecific antibody, of the effective amount of an anti-PD1/anti-LAG3antibody and administration of an additional therapeutic agent occurwithin about one month, or within about one, two or three weeks, orwithin about one, two, three, four, five, or six days, of each other.

Both the anti-CD20/anti-CD3 bispecific antibody and theanti-PD1/anti-LAG3 antibody as reported herein (and any additionaltherapeutic agent) can be administered by any suitable means, includingparenteral, intrapulmonary, and intranasal, and, if desired for localtreatment, intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Both the anti-CD20/anti-CD3 bispecific antibody and theanti-PD1/anti-LAG3 antibody as reported herein would be formulated,dosed, and administered in a fashion consistent with good medicalpractice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The antibodies need not be, but areoptionally formulated with one or more agents currently used to preventor treat the disorder in question. The effective amount of such otheragents depends on the amount of antibodies present in the formulation,the type of disorder or treatment, and other factors discussed above.These are generally used in the same dosages and with administrationroutes as described herein, or about from 1 to 99% of the dosagesdescribed herein, or in any dosage and by any route that isempirically/clinically determined to be appropriate.

A skilled artisan readily recognizes that in many cases the bispecificmolecule may not provide a cure but may only provide partial benefit. Insome embodiments, a physiological change having some benefit is alsoconsidered therapeutically beneficial. Thus, in some aspects, an amountof the bispecific antibody that provides a physiological change isconsidered an “effective amount” or a “therapeutically effectiveamount”.

Both the anti-CD20/anti-CD3 bispecific antibody and theanti-PD1/anti-LAG3 antibody as defined herein is suitably administeredto the patient at one time or over a series of treatments. Depending onthe type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g.0.1 mg/kg-10 mg/kg) of the bispecific antibody can be an initialcandidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. One typical daily dosage might range from about 1 μg/kg to 10mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment would generally be sustained until a desired suppressionof disease symptoms occurs. One exemplary dosage of theanti-CD20/anti-CD3 bispecific antibody would be in the range from about0.05 μg/kg to about 1000 μg/kg. For the anti-PD1/anti-LAG3 antibody, adose may also comprise from about 0.01 mg/kg body weight, about 0.05mg/kg body weight, about 2 mg/kg body weight, about 4 mg/kg body weight,about 10 mg/kg body weight, about 20 mg/kg body weight, about 30 mg/kgbody weight, about 40 mg/kg body weight, about 45 mg/kg body weight,about 50 mg/kg body weight, about 100 mg/kg body weight, about 200 mg/kgbody weight, about 300 mg/kg body weight, about 400 mg/kg body weight,about 500 mg/kg body weight, about 600 mg/kg body weight, about 800mg/kg body weight, about 1000 mg/kg body weight, top about 1200 mg/kgbody weight or more per administration, and any range derivable therein.In examples of a derivable range from the numbers listed herein, a rangeof about 5 mg/kg body weight to about 100 mg/kg body weight, about 0.05μg/kg body weight to about 500 mg/kg body weight etc., can beadministered, based on the numbers described above. In one aspect, theanti-CD20/anti-CD3 bispecific antibody may be administered to thepatient in a dose of from about 0.01 mg, from 2.5 mg, to about 10 mg orabout 20 mg or about 30 mg. Such doses may be administeredintermittently, e.g. every week or every three weeks (e.g. such that thepatient receives from about two to about twenty, or e.g. about six dosesof the fusion protein). An initial lower loading dose, followed by oneor more higher doses may be administered. However, other dosage regimensmay be useful. The progress of this therapy is easily monitored byconventional techniques and assays. In one aspect, theanti-PD1/anti-LAG3 antibody may be administered to the patient in a doseof from about 100 mg, about 200 mg, about 300 mg, about 400 mg, about500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg. about1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg orabout 1500 mg.

The bispecific antibodies comprising a first antigen binding domain thatspecifically binds to PD1 and a second antigen binding domain thatspecifically binds to LAG3 as defined herein will generally be used inan amount effective to achieve the intended purpose. For use to treat orprevent a disease condition, the bispecific antibodies of the invention,or pharmaceutical compositions thereof, are administered or applied in atherapeutically effective amount. Determination of a therapeuticallyeffective amount is well within the capabilities of those skilled in theart, especially in light of the detailed disclosure provided herein.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays, such as cell culture assays. Adose can then be formulated in animal models to achieve a circulatingconcentration range that includes the IC₅₀ as determined in cellculture. Such information can be used to more accurately determineuseful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data.

Dosage amount and interval may be adjusted individually to provideplasma levels of the bispecific antibody which are sufficient tomaintain therapeutic effect. Usual patient dosages for administration byinjection range from about 0.1 to 50 mg/kg/day, typically from about 0.5to 1 mg/kg/day. Therapeutically effective plasma levels may be achievedby administering multiple doses each day. Levels in plasma may bemeasured, for example, by HPLC.

In cases of local administration or selective uptake, the effectivelocal concentration of the bispecific antibody may not be related toplasma concentration. One skilled in the art will be able to optimizetherapeutically effective local dosages without undue experimentation.

A therapeutically effective dose of the bispecific antibodies describedherein will generally provide therapeutic benefit without causingsubstantial toxicity. Toxicity and therapeutic efficacy of a fusionprotein can be determined by standard pharmaceutical procedures in cellculture or experimental animals. Cell culture assays and animal studiescan be used to determine the LD₅₀ (the dose lethal to 50% of apopulation) and the ED₅₀ (the dose therapeutically effective in 50% of apopulation). The dose ratio between toxic and therapeutic effects is thetherapeutic index, which can be expressed as the ratio LD₅₀/ED₅₀.Bispecific antibodies that exhibit large therapeutic indices arepreferred. In one embodiment, the bispecific antibody according to thepresent invention exhibits a high therapeutic index. The data obtainedfrom cell culture assays and animal studies can be used in formulating arange of dosages suitable for use in humans. The dosage lies preferablywithin a range of circulating concentrations that include the ED50 withlittle or no toxicity. The dosage may vary within this range dependingupon a variety of factors, e.g., the dosage form employed, the route ofadministration utilized, the condition of the subject, and the like. Theexact formulation, route of administration and dosage can be chosen bythe individual physician in view of the patient's condition (see, e.g.,Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Ch.1, p. 1, incorporated herein by reference in its entirety).

The attending physician for patients treated with bispecific antibodiesof the invention would know how and when to terminate, interrupt, oradjust administration due to toxicity, organ dysfunction, and the like.Conversely, the attending physician would also know to adjust treatmentto higher levels if the clinical response were not adequate (precludingtoxicity). The magnitude of an administered dose in the management ofthe disorder of interest will vary with the severity of the condition tobe treated, with the route of administration, and the like. The severityof the condition may, for example, be evaluated, in part, by standardprognostic evaluation methods. Further, the dose and perhaps dosefrequency will also vary according to the age, body weight, and responseof the individual patient.

Such other agents are suitably present in combination in amounts thatare effective for the purpose intended. The effective amount of suchother agents depends on the amount of fusion protein used, the type ofdisorder or treatment, and other factors discussed above. The bispecificantibodies are generally used in the same dosages and withadministration routes as described herein, or about from 1 to 99% of thedosages described herein, or in any dosage and by any route that isempirically/clinically determined to be appropriate.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate compositions), and separate administration, in which case,administration of the bispecific antibody can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent and/or adjuvant.

H. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper that ispierceable by a hypodermic injection needle). At least one active agentin the composition is an anti-PD1/anti-LAG3 antibody as defined hereinbefore.

The label or package insert indicates that the composition is used fortreating the condition of choice. Moreover, the article of manufacturemay comprise (a) a first container with a composition contained therein,wherein the composition comprises an anti-CD2W/anti-CD3 bispecificantibody and (b) a second container with a composition containedtherein, wherein the composition comprises an anti-PD1/anti-LAG3antibody. The article of manufacture in this embodiment of the inventionmay further comprise a package insert indicating that the compositionscan be used to treat a particular condition.

Alternatively, or additionally, the article of manufacture may furthercomprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

TABLE C (Sequences): SEQ ID NO: Name Sequence 1heavy chain HVR-H1, PD1-0103 GFSFSSY 2 heavy chain HVR-H2, PD1-0103 GGR3 heavy chain HVR-H3, PD1-0103 TGRVYFALD 4 light chain HVR-L1, PD1-0103SESVDTSDNSF 5 light chain HVR-L2, PD1-0103 RSS 6light chain HVR-L3, PD1-0103 NYDVPW 7 heavy chain variable domain VH,EVILVESGGGLVKPGGSLKLSCAASGFSFSSYTM PD1-0103SWVRQTPEKRLDWVATISGGGRDIYYPDSVKGRF TISRDNAKNTLYLEMSSLMSEDTALYYCVLLTGRVYFALDSWGQGTSVTVSS 8 light chain variable domain VL,KIVLTQSPASLPVSLGQRATISCRASESVDTSDN PD1-0103SFIHWYQQRPGQSPKLLIYRSSTLESGVPARFSG SGSRTDFTLTIDPVEADDVATYYCQQNYDVPWTFGGGTKLEIK 9 humanized variant -heavy chainEVQLLESGGGLVQPGGSLRLSCAASGFSFSSYTM variable domain VH of PD1-SWVRQAPGKGLEWVATISGGGRDIYYPDSVKGRF 0103_01 (PD1 0376)TISRDNSKNTLYLQMNSLRAEDTAVYYCVLLTGR VYFALDSWGQGTLVTVSS 10humanized variant -light chain DIVMTQSPDSLAVSLGERATINCKASESVDTSDNvariable domain VL of PD1- SFIHWYQQKPGQSPKLLIYRSSTLESGVPDRESG0103_01 (PD1 0376) SGSGTDFTLTISSLQAEDVAVYYCQQNYDVPWTF GQGTKVEIK 11heavy chain HVR-H1, DYTMN aLAG3(0414) 12 heavy chain HVR-H2,VISWDGGGTY YTDSVKG aLAG3(0414) 13 heavy chain HVR-H3, GLTDTTLYGS DYaLAG3(0414) 14 light chain HVR-L1, aLAG3(0414) RASQSISSYL N 15light chain HVR-L2, aLAG3(0414) AASTLQS 16light chain HVR-L3, aLAG3(0414) QQTYSSPLT 17heavy chain variable domain VH, EVQLLESGGGLVQPGGSLRLSCAASGFIFDDYTMaLAG3(0414) NWVRQAPGKGLEWVAVISWDGGGTYYTDSVKGRFTISRDDFKNTLYLQMNSLRAEDTAVYYCAKGLTD TTLYGSDYWGQGTLVTVSS 18light chain variable domain VL, DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNaLAG3(0414) WYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYSSPLTFGGGT KVEIK 19 heavy chain HVR-H1, DYAMSaLAG3(0416) 20 heavy chain HVR-H2, GIDNSGYYTY YTDSVKG aLAG3(0416) 21heavy chain HVR-H3, THSGLIVNDA FDI aLAG3(0416) 22light chain HVR-L1, aLAG3(0416) RASQSISSYL N 23light chain HVR-L2, aLAG3(0416) DASSLES 24light chain HVR-L3, aLAG3(0416) QQSYSTPLT 25heavy chain variable domain VH, EVQLVESGGGLVQPGGSLRLACAASGFTFSDYAMaLAG3(0416) SWVRQAPGKGLEWVSGIDNSGYYTYYTDSVKGRFTISRDDVKNTLYLQMNSLRAEDTAVYLCTKTHSG LIVNDAFDIWGQGTMVTVSS 26light chain variable domain VL, DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNaLAG3(0416) WYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDATLTISSLQPEDFATYYCQQSYSTPLTFGGGT KVEIK 27heavy chain variable domain VH, QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWBMS-986016 NWIRQPPGKGLEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDY EYNWFDPWGQGTLVTVSS 28light chain variable domain VL EIVLTQSPATLSLSPGERATLSCRASQSISSYLABMS-986016 WYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGT NLEIK 29heavy chain variable domain VH, QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWMDX25F7 (25F7) NWIRQPPGKGLEWIGEINHNGNTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDY EYNWFDPWGQGTLVTVSS 30light chain variable domain VL, EIVLTQSPATLSLSPGERATLSCRASQSISSYLAMDX25F7 (25F7) WYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGT NLEIK 31heavy chain variable domain VH, QVQLVQSGAEVKKPGASVKVSCKASGFTLTNYGMhumanized BAP050 (LAG525) NWVRQARGQRLEWIGWINTDTGEPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARNPPY YYGTNNAEAMDYWGQGTTVTVSS 32light chain variable domain VL, DIQMTQSPSSLSASVGDRVTITCSSSQDISNYLNhumanized BAP050 (LAG525) WYLQKPGQSPQLLIYYTSTLHLGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYYNLPWTFGQGT KVEIK 33heavy chain variable domain VH, QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMMDX26H10 (26H10) HWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREWAV ASWDYGMDVWGQGTTVTVSS 34light chain variable domain VL, EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLMDX26H10 (26H10) AWYQQKPGQAPRLLIYGASSRATGIPDRESGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPFTFG PGTKVDIK 35heavy chain 1 of 1 + 1 PD1/LAG3 DIVMTQSPDSLAVSLGERATINCKASESVDTSDN0927 based on PD1(0376) SFIHWYQQKPGQSPKLLIYRSSTLESGVPDRESGSGSGTDFTLTISSLQAEDVAVYYCQQNYDVPWTF GQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKENWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 36 light chain 1 of 1 + 1 PD1/LAG3EVQLLESGGGLVQPGGSLRLSCAASGFSFSSYTM 0927 based on PD1(0376)SWVRQAPGKGLEWVATISGGGRDIYYPDSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCVLLTGRVYFALDSWGQGTLVTVSSASVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSENRGEC 37heavy chain 2 of 1 + 1 PD1/LAG3 EVQLLESGGGLVQPGGSLRLSCAASGFIFDDYTM0927 based on aLAG3(0414) NWVRQAPGKGLEWVAVISWDGGGTYYTDSVKGRFTISRDDFKNTLYLQMNSLRAEDTAVYYCAKGLTD TTLYGSDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEA AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKENWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSP 38light chain 2 of 1 + 1 PD1/LAG3 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLN0927 based on aLAG3(0414) WYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYSSPLTFGGGT KVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENRGEC 39 heavy chain 2 of 1 + 1 PD1/LAG3EVQLVESGGGLVQPGGSLRLACAASGFTFSDYAM 0799 based on aLAG3(0416)SWVRQAPGKGLEWVSGIDNSGYYTYYTDSVKGRF TISRDDVKNTLYLQMNSLRAEDTAVYLCTKTHSGLIVNDAFDIWGQGTMVTVSSASTKGPSVFPLAPS SKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKT ISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 40 light chain 2 of 1 + 1 PD1/LAG3DIQLTQSPSSLSASVGDRVTITCRASQSISSYLN 0799 based on aLAG3(0416)WYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSG TDATLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSENRGEC 41 CD3-HCDR1 TYAMN 42CD3-HCDR2 RIRSKYNNYATYYADSVKG 43 CD3-HCDR3 HGNFGNSYVSWFAY 44 CD3-LCDR1GSSTGAVTTSNYAN 45 CD3-LCDR2 GTNKRAP 46 CD3-LCDR3 ALWYSNLWV 47 CD3 VHEVQLLESGGGLVQPGGSLRLSCAASGFTESTYAM NWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHG NFGNSYVSWFAYWGQGTLVTVSS 48 CD3 VLQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNY ANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGG GTKLTVL 49 CD20-HCDR1 YSWIN 50CD20-HCDR2 RIFPGDGDTDYNGKFKG 51 CD20-HCDR3 NVFDGYWLVY 52 CD20-LCDR1RSSKSLLHSNGITYLY 53 CD20-LCDR2 QMSNLVS 54 CD20-LCDR3 AQNLELPYT 55CD20 VH QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRV TITADKSTSTAYMELSSLRSEDTAVYYCARNVEDGYWLVYWGQGTLVTVSS 56 CD20 VL DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLVSGVPDRES GSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIK 57 CD20 VH-CHI(EE)-CD3 VL-CHI-QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWI Fc (knob, P329G LALA)NWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRV TITADKSTSTAYMELSSLRSEDTAVYYCARNVEDGYWLVYWGQGTLVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDEKVEPKSCDGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQ EKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLT VLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKENWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 58 CD20 VH-CHI(EE)-Fc (hole,QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWI P329G LALA)NWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRV TITADKSTSTAYMELSSLRSEDTAVYYCARNVEDGYWLVYWGQGTLVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISK AKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL VSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 59 CD20 VL-CL(RK) DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLVSGVPDRES GSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIKRTVAAPSVFIFPPSDRKLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSENRGEC 60 CD3 VH-CLEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAM NWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHG NFGNSYVSWFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 61 CD20 UniProt accession No. P11836 62Obinutuzumab heavy chain QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRV TITADKSTSTAYMELSSLRSEDTAVYYCARNVEDGYWLVYWGQGTLVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 63 Obinutuzumab light chain DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLVSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC 64murine anti-CD20 B-Ly1 GPELVKPGASVKISCKASGYAFSYSWMNWVKLRP VHGQGLEWIGRIFPGDGDTDYNGKFKGKATLTADKS SNTAYMQLTSLTSVDSAVYLCARNVEDGYWLVYWGQGTLVTVSA 65 murine anti-CD20 B-Ly1 NPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKVL PGQSPQLLIYQMSNLVSGVPDRESSSGSGTDFTL RISRVEAEDVGVYYCAQNLELPYTFGGGTKLEIKR 66 human CD3ε UniProt accession no. P07766 67 cynomolgus CD3εNCBI GenBank no. BAB71849.1 68 human PD-1 UniProt accession no. Q1511669 exemplary human LAG3 sequence VPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVT(without signal sequence) WQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRG DFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRPA SVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPT PLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCH IHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQE AQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHLLLFLILGVLSLLLLVTGAFG FHLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPEPEPEPEPEPEPEPEPEQL 70 human LAG3 Extracellular DomainVPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVT (ECD)WQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRP RRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRL RLGQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVS PMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTP PGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLL CEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQ RSGRAPGALPAGHL 71 Peptide linker G₄SGGGGS 72 Peptide linker (G₄S)₂ GGGGSGGGGS 73 Peptide linker (G₄S)₃GGGGSGGGGSGGGGS 74 Peptide linker (G₄S)₄ GGGGSGGGGSGGGGSGGGGS 75Pembrolizumab heavy chain QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRV TLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSR STSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 76 Pembrolizumab light chain EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSG SGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSENRGEC 77Nivolumab heavy chain QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRF TISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN TKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKS RWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 78Nivolumab light chain EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSG TDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSENRGEC 79 Anti-Lag3 heavy chainQMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNV DWVRQARGQRLEWIGDINPNDGGTIYAQKFQERVTITVDKSTSTAYMELSSLRSEDTAVYYCARNYRW FGAMDHWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSP 80 Anti-Lag3 light chainDIVMTQTPLSLSVTPGQPASISCKASQSLDYEGD SDMNWYLQKPGQPPQLLIYGASNLESGVPDRESGSGSGTDFTLKISRVEAEDVGVYYCQQSTEDPRTF GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 81 Anti-Lag3 heavy chain variableQMQLVQSGPEVKKPGTSVKVSCKASGYTFTDYNV domain VHDWVRQARGQRLEWIGDINPNDGGTIYAQKFQERV TITVDKSTSTAYMELSSLRSEDTAVYYCARNYRWFGAMDHWGQGTTVTVSS 82 Anti-Lag3 light chain variableDIVMTQTPLSLSVTPGQPASISCKASQSLDYEGD domain VLSDMNWYLQKPGQPPQLLIYGASNLESGVPDRESG SGSGTDFTLKISRVEAEDVGVYYCQQSTEDPRTFGGGTKVEIK 83 CD3 (40G5c)-HCDR1 NYYIH 84 CD3 (40G5c)-HCDR2WIYPGDGNTKYNEKFKG 85 CD3 (40G5c)-HCDR3 DSYSNYYFDY 86 CD3 (40G5c)-LCDR1KSSQSLLNSRTRKNYLA 87 CD3 (40G5c)-LCDR2 WASTRES 88 CD3 (40G5c)-LCDR3TQSFILRT 89 CD3 (40G5c) VH EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQGLEWIGWIYPGDGNTKYNEKFKGRA TLTADTSTSTAYLELSSLRSEDTAVYYCARDSYSNYYFDYWGQGTLVTVSS 90 CD3 (40G5c) VL DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRF SGSGSGTDFTLTISSLQAEDVAVYYCTQSFILRTFGQGTKVEIK 91 CD20 (2H7.v16)-HCDR1 GYTFTSYNMH 92 CD20 (2H7.v16)-HCDR2AIYPGNGDTSYNQKFKG 93 CD20 (2H7.v16)-HCDR3 VVYYSNSYWYFD V 94CD20 (2H7.v16)-LCDR1 RASSSVSYMH 95 CD20 (2H7.v16)-LCDR2 APSNLAS 96CD20 (2H7.v16)-LCDR3 QQWSENPPT 97 CD20 (2H7.v16) VHEVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNM HWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYY SNSYWYFDVWGQGTLVTVSS 98CD20 (2H7.v16) VL DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIK 99 CD3 (40G5c) light chain DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRF SGSGSGTDFTLTISSLQAEDVAVYYCTQSFILRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC 100CD3 (40G5c) heavy chain EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQGLEWIGWIYPGDGNTKYNEKFKGRA TLTADTSTSTAYLELSSLRSEDTAVYYCARDSYSNYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL VSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 101 CD20 (2H7.v16) light chain DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQWSENPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC 102CD20 (2H7.v16) heavy chain EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRF TISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 103 KIEELE (part of LAG intracellular KIEELE domain)

General information regarding the nucleotide sequences of humanimmunoglobulins light and heavy chains is given in: Kabat, E. A., etal., Sequences of Proteins of Immunological Interest, 5th ed., PublicHealth Service, National Institutes of Health, Bethesda, MD (1991).Amino acids of antibody chains are numbered and referred to according tothe numbering systems according to Kabat (Kabat, E. A., et al.,Sequences of Proteins of Immunological Interest, 5th ed., Public HealthService, National Institutes of Health, Bethesda, MD (1991)) as definedabove.

ASPECTS OF THE INVENTION

In the following, some of the aspects of the invention are listed.

-   -   1. An anti-CD20/anti-CD3 bispecific antibody for use in a method        of treating CD20-expressing cancer, wherein the        anti-CD20/anti-CD3 bispecific antibody is used in combination        with an anti-PD1/anti-LAG3 bispecific antibody.    -   2. The anti-CD20/anti-CD3 bispecific antibody for use in a        method of paragraph (para) 1, wherein the anti-CD20/anti-CD3        bispecific antibody and the anti-PD1/anti-LAG3 bispecific        antibody are administered together in a single composition or        administered separately in two or more different compositions.    -   3. The anti-CD20/anti-CD3 bispecific antibody for use in a        method of paras 1 or 2, wherein the anti-PD1/anti-LAG3        bispecific antibody comprises a Fc domain that is an IgG Fc        domain, particularly an IgG1 Fc domain or an IgG4 Fc domain, and        wherein the Fc domain comprises one or more amino acid        substitution that reduces binding to an Fc receptor, in        particular towards Fcγ receptor.    -   4. The anti-CD20/anti-CD3 bispecific antibody for use in a        method of any one of paras 1 to 3, wherein the        anti-PD1/anti-LAG3 bispecific antibody comprises a Fc domain of        human IgG1 subclass with the amino acid mutations L234A, L235A        and P329G (numbering according to Kabat EU index).    -   5. The anti-CD20/anti-CD3 bispecific antibody for use in a        method of any one of paras 1 to 4, wherein the        anti-PD1/anti-LAG3 bispecific antibody comprises a first antigen        binding domain that specifically binds to programmed cell death        protein 1 (PD1) and a second antigen binding domain that        specifically binds to Lymphocyte activation gene-3 (LAG3),        wherein a first antigen binding domain specifically binding to        PD1 comprises a VH domain comprising    -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1,    -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2,        and    -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID NO:3;        and        a VL domain comprising    -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:4;    -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5,        and    -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6.    -   6. The anti-CD20/anti-CD3 bispecific antibody for use in a        method of any one of paras 1 to 4, wherein the        anti-PD1/anti-LAG3 bispecific antibody comprises a second        antigen binding domain that specifically binds to LAG3        comprising    -   (a) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:11,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:12, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:13; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:14,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:15, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO: 16; or    -   (b) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:19,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:20, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:21; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:22,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:23, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:24.    -   7. The anti-CD20/anti-CD3 bispecific antibody for use in a        method of any one of paras 1 to 6, wherein the        anti-PD1/anti-LAG3 bispecific antibody comprises a first        antigen-binding domain specifically binding to PD1 comprising        the VH domain comprising the amino acid sequence of SEQ ID NO: 9        and the VL domain comprising the amino acid sequence of SEQ ID        NO: 10.    -   8. The anti-CD20/anti-CD3 bispecific antibody for use in a        method of any one of paras 1 to 7, wherein the        anti-PD1/anti-LAG3 bispecific antibody comprises a second        antigen-binding domain specifically binding to LAG3 comprising        -   (a) a VH domain comprising the amino acid sequence of SEQ ID            NO: 17 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 18, or        -   (b) a VH domain comprising the amino acid sequence of SEQ ID            NO: 25 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 26.    -   9. The anti-CD20/anti-CD3 bispecific antibody for use in a        method of any one of paras 1 to 5 or 7, wherein the        anti-PD1/anti-LAG3 bispecific antibody comprises a second        antigen-binding domain specifically binding to LAG3 comprising        -   (a) a VH domain comprising the amino acid sequence of SEQ ID            NO: 27 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 28, or        -   (b) a VH domain comprising the amino acid sequence of SEQ ID            NO: 29 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 30, or        -   (c) a VH domain comprising the amino acid sequence of SEQ ID            NO: 31 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 32, or        -   (d) a VH domain comprising the amino acid sequence of SEQ ID            NO: 33 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 34.    -   10. The anti-CD20/anti-CD3 bispecific antibody for use in a        method of any one of paras 1 to 9, wherein the        anti-PD1/anti-LAG3 bispecific antibody comprises        -   a first antigen binding domain specifically binding to PD1            comprising a VH domain comprising the amino acid sequence of            SEQ ID NO: 9 and a VL domain comprising the amino acid            sequence of SEQ ID NO: 10,        -   and a second antigen binding domain specifically binding to            LAG3 comprising a VH domain comprising the amino acid            sequence of SEQ ID NO: 17 and a VL domain comprising the            amino acid sequence of SEQ ID NO: 18.    -   11. The anti-CD20/anti-CD3 bispecific antibody for use in a        method of any one of paras 1 to 10, wherein the        anti-PD1/anti-LAG3 bispecific antibody comprises a Fab fragment        specifically binding to PD1 and a Fab fragment specifically        binding to LAG3.    -   12. The anti-CD20/anti-CD3 bispecific antibody for use in a        method of any one of paras 1 to 11, wherein the        anti-PD1/anti-LAG3 bispecific antibody comprises a Fab fragment        specifically binding to PD1, wherein the variable domains VL and        VH are replaced by each other so that VL is part of the heavy        chain and VH is part of the light chain.    -   13. The anti-CD20/anti-CD3 bispecific antibody for use in a        method of any one of paras 1 to 12, wherein the        anti-PD1/anti-LAG3 bispecific antibody comprises monovalent        binding to PD-1 and monovalent binding to LAG3.    -   14. The anti-CD20/anti-CD3 bispecific antibody for use in a        method of any one of paras 1 to 13, wherein the        anti-PD1/anti-LAG3 bispecific antibody comprises    -   (a) a first heavy chain comprising an amino acid sequence of SEQ        ID NO: 35, a first light chain comprising an amino acid sequence        of SEQ ID NO: 36, a second heavy chain comprising an amino acid        sequence of SEQ ID NO: 37, and a second light chain comprising        an amino acid sequence of SEQ ID NO:38, or    -   (b) a first heavy chain comprising an amino acid sequence of SEQ        ID NO: 35, a first light chain comprising an amino acid sequence        of SEQ ID NO: 36, a second heavy chain comprising an amino acid        sequence of SEQ ID NO: 39, and a second light chain comprising        an amino acid sequence of SEQ ID NO:40.    -   15. The anti-CD20/anti-CD3 bispecific antibody for use in a        method of any one of paras 1 to 14, wherein the        anti-PD1/anti-LAG3 bispecific antibody comprises a first heavy        chain comprising an amino acid sequence of SEQ ID NO: 35, a        first light chain comprising an amino acid sequence of SEQ ID        NO: 36, a second heavy chain comprising an amino acid sequence        of SEQ ID NO: 37, and a second light chain comprising an amino        acid sequence of SEQ ID NO:38.    -   16. The anti-CD20/anti-CD3 bispecific antibody for use in a        method of any one of paras 1 to 15, wherein the        anti-CD20/anti-CD3 bispecific antibody comprises a first antigen        binding domain comprising a heavy chain variable region        (V_(H)CD3) and a light chain variable region (V_(L)CD3), and a        second antigen binding domain comprising a heavy chain variable        region (V_(H)CD20) and a light chain variable region        (V_(L)CD20).    -   17. The anti-CD20/anti-CD3 bispecific antibody for use in a        method of any one of paras 1 to 16, wherein the first antigen        binding domain comprises a heavy chain variable region (VHCD3)        comprising CDR-H1 sequence of SEQ ID NO:41, CDR-H2 sequence of        SEQ ID NO:42, and CDR-H3 sequence of SEQ ID NO:43; and/or a        light chain variable region (V_(L)CD3) comprising CDR-L1        sequence of SEQ ID NO:44, CDR-L2 sequence of SEQ ID NO:45, and        CDR-L3 sequence of SEQ ID NO:46.    -   18. The anti-CD20/anti-CD3 bispecific antibody for use in a        method of any one of paras 1 to 17, wherein the first antigen        binding domain comprises a heavy chain variable region (VHCD3)        comprising the amino acid sequence of SEQ ID NO:47 and/or a        light chain variable region (V_(L)CD3) comprising the amino acid        sequence of SEQ ID NO:48.    -   19. The anti-CD20/anti-CD3 bispecific antibody for use in a        method of any one of paras 1 to 18, wherein the second antigen        binding domain comprises a heavy chain variable region        (V_(H)CD20) comprising CDR-H1 sequence of SEQ ID NO:49, CDR-H2        sequence of SEQ ID NO:50, and CDR-H3 sequence of SEQ ID NO:51,        and/or a light chain variable region (V_(L)CD20) comprising        CDR-L1 sequence of SEQ ID NO:52, CDR-L2 sequence of SEQ ID        NO:53, and CDR-L3 sequence of SEQ ID NO:54.    -   20. The anti-CD20/anti-CD3 bispecific antibody for use in a        method of any one of paras 1 to 19, wherein the second antigen        binding domain comprises a heavy chain variable region        (V_(H)CD20) comprising the amino acid sequence of SEQ ID NO:55        and/or a light chain variable region (V_(L)CD20) comprising the        amino acid sequence of SEQ ID NO: 56.    -   21. The anti-CD20/anti-CD3 bispecific antibody for use in a        method of any one of paras 1 to 20, wherein the        anti-CD20/anti-CD3 bispecific antibody comprises a third antigen        binding domain that binds to CD20.    -   22. The anti-CD20/anti-CD3 bispecific antibody for use in a        method of any one of paras 1 to 21, wherein the        anti-CD20/anti-CD3 bispecific antibody comprises an Fc domain        comprising one or more amino acid substitutions that reduce        binding to an Fc receptor and/or effector function.    -   23. The anti-CD20/anti-CD3 bispecific antibody for use in a        method of any one of paras 1 to 22, wherein the        anti-CD20/anti-CD3 bispecific antibody is used in combination        with anti-PD1/anti-LAG3 bispecific antibody and wherein the        combination is for administration at intervals from about one        week to three weeks.    -   24. The anti-CD20/anti-CD3 bispecific antibody for use in a        method of any one of paras 1 to 23, wherein a pretreatment with        an Type II anti-CD20 antibody, preferably obinutuzumab, is        performed prior to the combination treatment, wherein the period        of time between the pretreatment and the combination treatment        is sufficient for the reduction of B-cells in the individual in        response to the Type II anti-CD20 antibody, preferably        obinutuzumab.    -   25. A pharmaceutical composition comprising a combination of an        anti-CD20/anti-CD3 bispecific antibody and an anti-PD1/anti-LAG3        bispecific antibody for use in the combined, sequential or        simultaneous, treatment of a disease, in particular CD20        expressing cancer.    -   26. A pharmaceutical composition comprising an        anti-CD20/anti-CD3 bispecific antibody and a pharmaceutically        acceptable carrier, and a second medicament comprising an        anti-PD1/anti-LAG3 bispecific antibody.    -   27. The pharmaceutical composition of para 26 for use in the        treatment of a CD20 expressing cancer, in particular a        hematological cancer selected from the group consisting of        Non-Hodgkin lymphoma (NHL), acute lymphocytic leukemia (ALL),        chronic lymphocytic leukemia (CLL), diffuse large B-cell        lymphoma (DLBCL), follicular lymphoma (FL), mantle-cell lymphoma        (MCL), marginal zone lymphoma (MZL), Multiple myeloma (MM) and        Hodgkin lymphoma (HL).    -   28. Use of a combination of an anti-CD20/anti-CD3 bispecific        antibody and an anti-PD1/anti-LAG3 bispecific antibody in the        manufacture of a medicament for treating a CD20 expressing        cancer.    -   29. A method for treating a CD20 expressing cancer in a subject        comprising administering to the subject an effective amount of        an anti-CD20/anti-CD3 antibody and an effective amount of an        anti-PD1/anti-LAG3 bispecific antibody.    -   30. The method of para 29, wherein the anti-CD20/anti-CD3        bispecific antibody and the anti-PD1/anti-LAG3 bispecific        antibody are administered together in a single composition or        administered separately in two or more different compositions.    -   31. The method of paras 29 or 30, wherein the anti-CD20/anti-CD3        bispecific antibody and the anti-PD1/anti-LAG3 bispecific        antibody are administered intravenously or subcutaneously.    -   32. The method of any one of paras 29 to 31, wherein the        anti-CD20/anti-CD3 bispecific antibody is administered        concurrently with, prior to, or subsequently to the        anti-PD1/anti-LAG3 bispecific antibody.

EXAMPLES

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook etal., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, New York, 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions. General information regarding the nucleotide sequences ofhuman immunoglobulin light and heavy chains is given in: Kabat, E. A. etal., (1991) Sequences of Proteins of Immunological Interest, Fifth Ed.,NIH Publication No 91-3242.

DNA Sequencing

DNA sequences were determined by double strand sequencing.

Gene Synthesis

Desired gene segments were either generated by PCR using appropriatetemplates or were synthesized by Geneart AG (Regensburg, Germany) fromsynthetic oligonucleotides and PCR products by automated gene synthesis.In cases where no exact gene sequence was available, oligonucleotideprimers were designed based on sequences from closest homologues and thegenes were isolated by RT-PCR from RNA originating from the appropriatetissue. The gene segments flanked by singular restriction endonucleasecleavage sites were cloned into standard cloning/sequencing vectors. Theplasmid DNA was purified from transformed bacteria and concentrationdetermined by UV spectroscopy. The DNA sequence of the subcloned genefragments was confirmed by DNA sequencing. Gene segments were designedwith suitable restriction sites to allow sub-cloning into the respectiveexpression vectors. All constructs were designed with a 5′-end DNAsequence coding for a leader peptide which targets proteins forsecretion in eukaryotic cells.

Cell Culture Techniques

Standard cell culture techniques were used as described in CurrentProtocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M., Harford,J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.), John Wiley &Sons, Inc.

Protein Purification

Proteins were purified from filtered cell culture supernatants referringto standard protocols. In brief, antibodies were applied to a Protein ASepharose column (GE healthcare) and washed with PBS. Elution ofantibodies was achieved at pH 2.8 followed by immediate neutralizationof the sample. Aggregated protein was separated from monomericantibodies by size exclusion chromatography (Superdex 200, GEHealthcare) in PBS or in 20 mM Histidine, 150 mM NaCl pH 6.0. Monomericantibody fractions were pooled, concentrated (if required) using e.g., aMILLIPORE Amicon Ultra (30 MWCO) centrifugal concentrator, frozen andstored at −20° C. or −80° C. Part of the samples were provided forsubsequent protein analytics and analytical characterization e.g. bySDS-PAGE, size exclusion chromatography (SEC) or mass spectrometry.

SDS-PAGE

The NuPAGE® Pre-Cast gel system (Invitrogen) was used according to themanufacturer's instruction. In particular, 10% or 4-12% NuPAGE® Novex®Bis-TRIS Pre-Cast gels (pH 6.4) and a NuPAGE® MES (reduced gels, withNuPAGE® Antioxidant running buffer additive) or MOPS (non-reduced gels)running buffer was used.

Analytical Size Exclusion Chromatography

Size exclusion chromatography (SEC) for the determination of theaggregation and oligomeric state of antibodies was performed by HPLCchromatography. Briefly, Protein A purified antibodies were applied to aTosoh TSKgel G3000SW column in 300 mM NaCl, 50 mM KH₂PO₄/K₂HPO₄, pH 7.5on an Agilent HPLC 1100 system or to a Superdex 200 column (GEHealthcare) in 2×PBS on a Dionex HPLC-System. The eluted protein wasquantified by UV absorbance and integration of peak areas. BioRad GelFiltration Standard 151-1901 served as a standard.

Determination of Binding and Binding Affinity of MultispecificAntibodies to the Respective Antigens Using Surface Plasmon Resonance(SPR) (BIACORE)

Binding of the generated antibodies to the respective antigens isinvestigated by surface plasmon resonance using a BIACORE instrument (GEHealthcare Biosciences AB, Uppsala, Sweden). Briefly, for affinitymeasurements Goat-Anti-Human IgG, JIR 109-005-098 antibodies areimmobilized on a CM5 chip via amine coupling for presentation of theantibodies against the respective antigen. Binding is measured in HBSbuffer (HBS-P (10 mM HEPES, 150 mM NaCl, 0.005% Tween 20, ph 7.4), 25°C. (or alternatively at 37° C.). Antigen (R&D Systems or in housepurified) was added in various concentrations in solution. Associationwas measured by an antigen injection of 80 seconds to 3 minutes;dissociation was measured by washing the chip surface with HBS bufferfor 3-10 minutes and a KD value was estimated using a 1:1 Langmuirbinding model. Negative control data (e.g. buffer curves) are subtractedfrom sample curves for correction of system intrinsic baseline drift andfor noise signal reduction. The respective Biacore Evaluation Softwareis used for analysis of sensorgrams and for calculation of affinitydata.

Example 1 Preparation, Purification and Characterization of T-CellBispecific (TCB) Antibodies

TCB molecules have been prepared according to the methods described inWO 2016/020309 A1.

The anti-CD20/anti-CD3 bispecific antibody (CD20 CD3 TCB or CD20 TCB)used in the experiments corresponds to molecule B as described inExample 1 of WO 2016/020309 A1. Molecule B is a “2+1 IgG CrossFab”antibody and is comprised of two different heavy chains and twodifferent light chains. Point mutations in the CH3 domain (“knobs intoholes”) were introduced to promote the assembly of the two differentheavy chains. The Pro329Gly, Leu234Ala and Leu235Ala mutations wereintroduced in the constant region of the knob and hole heavy chains toabrogate binding to Fc gamma receptors according to the method describedin WO 2012/130831. Exchange of the VH and VL domains in the CD3 bindingFab and point mutations in the CH and CL domains in the CD20 binding Fabwere made in order to promote the correct assembly of the two differentlight chains. 2+1 means that the molecule has two antigen bindingdomains specific for CD20 and one antigen binding domain specific forCD3.

CD20 TCB comprises the amino acid sequences of SEQ ID NO:57, SEQ IDNO:58, SEQ ID NO:59 and SEQ ID NO:60. A schematic scheme of thebispecific antibody in 2+1 format is shown in FIG. 1B.

The molecule is further characterized in Example 1 of WO 2016/020309 A1.

Example 2 Preparation, Purification and Characterization of BispecificAnti-PD1/Anti-LAG3 Antibodies

Bispecific antibodies which bind to human PD1 and human LAG3 with VH/VLdomain exchange/replacement (CrossMAb^(Vh-VL)) in one binding arm weregenerated as described in Example 10.1 of WO 2018/185043. Thepreparation of multispecific 1+1 CrossMAb^(Vh-Vl)-antibodies is alsodescribed in WO 2009/080252. The bispecific antibodies were expressedusing expression plasmids containing the nucleic acids encoding theamino acid sequences depicted in Table 1. A schematic structure of the1+1 CrossMAb^(Vh-Vl) bispecific antibodies is shown in FIG. 1A.

TABLE 1 Amino acid sequences of light chains (LC) and heavy chains (HC),with VH/VL domain exchange/replacement (1 + 1 CrossMAb^(Vh-Vl)) 1 + 1Antibody HC1 HC2 LC1 LC2 PD1/LAG3 0799 SEQ ID SEQ ID SEQ ID SEQ IDPD1(0376)/ NO: 35 NO: 39 NO: 36 NO: 40 aLAG3(0416) PD1/LAG3 0927 SEQ IDSEQ ID SEQ ID SEQ ID PD1(0376)/ NO: 35 NO: 37 NO: 36 NO: 38 aLAG3(0414)

For all constructs knobs into holes heterodimerization technology wasused with a typical knob (T366W) substitution in the first CH3 domainand the corresponding hole substitutions (T366S, L368A and Y410V) in thesecond CH3 domain (as well as two additional introduced cysteineresidues S354C/Y349′C) (contained in the respective corresponding heavychain (HC) sequences depicted above). The Pro329Gly, Leu234Ala andLeu235Ala mutations were introduced in the constant region of the knoband hole heavy chains to abrogate binding to Fc gamma receptorsaccording to the method described in WO 2012/130831. In order to improvecorrect pairing, amino acid substitutions were additionally introducedin the CH and CL domain of the conventional Fab (charged variants).

The bispecific antibodies expressed as above were purified from thesupernatant by a combination of Protein A affinity chromatography andsize exclusion chromatography. The obtained products were characterizedfor identity by mass spectrometry and analytical properties such aspurity by SDS-PAGE, monomer content and stability.

The parental PD1 antibody PD1(0376) IgG1 used for comparison comprisesthe VH domain comprising the amino acid sequence of SEQ ID NO:9 and theVL domain comprising the amino acid sequence of SEQ ID NO:10.

Example 3 Effect of PD-1/LAG-3 Bispecific Antibodies in Combination withCD20 CD3 TCB on Cytotoxic Granzyme B Release by Human CD4 T CellsCocultured with a B Cell-Lymphoblatoid Cell Line (ARH77)

To investigate the combinability of the PD-1/LAG-3 bispecific antibodieswith CD20-TCB, we developed an assay in which freshly purified CD4 Tcells are co-cultured for 5 days in presence of an EBV-immortalizedB-cell lymphoblastoid tumor cell line (ARH77). We chose the ARH77 cellline because of its intermediate expression levels of PD-1 ligand,PD-L1, and high levels of LAG-3 ligand MHC-II, allowing for theassessment of the contribution of LAG-3 blockade in addition to PD-1.

CD4 T cells were enriched via a microbead kit (Miltenyi Biotec) from 108PBMCs obtained from 5 healthy donors. Prior culture, CD4 T cells werelabeled with 5 μM of Carboxy-Fluorescein-Succinimidyl Esther (CFSE). 105CD4 T cells were then plated in a 96 well plate together with the B cellline (5:1) in presence or absence of blocking anti-PD1 antibodies(either parental anti-PD-1, nivolumab or pembrolizumab) or thePD-1/LAG-3 bispecific antibody PD1/LAG3 0927 (PD1-LAG3 BsAb) atconcentrations between 10⁻⁷ and 10 μg/ml and a fixed concentration ofCD20-TCB (66 μM). Five days later, for the last five hours ofincubation, we added Golgi-plug and Golgi-stop to block proteintransportation and allow intracellular accumulation of the cytokines.

Interestingly, we observed a dose dependent effect of PD-1 blockingantibodies in combination with CD20-TCB on CD4 T cell secretion ofGranzyme B (see FIG. 2 ). However, equimolar PD1-LAG3 BsAb was morepotent and efficacious (E_(max)) than the PD-1 blocking antibodies inincreasing in a dose dependent fashion the Granzyme B secreted by CD4 Tcells, making it a suitable combination partner for CD20-TCB. Thecorresponding EC50 values are shown in Table 2 below:

TABLE 2 Granzyme B secreted by CD4 T cells in coculture with ARH77 whenplated with PD1-LAG3 BsAb or blocking anti- PD1 antibodies incombination with CD20 CD3 TCB Molecule EC₅₀ pembrolizumab n.d. nivolumab4.039 Parental aPD-1 antibody PD1(0376) 0.3318 PD1-LAG3 BsAb (PD1/LAG30927) 0.2162

Example 4 Potent Anti-Tumor Effect of the Combination Therapy ofPD1/LAG3 Bispecific Antibodies and CD20 CD3 TCB In Vivo in the WSU-DLCL2Graft Model in Humanized NSG Mice

The anti-tumour activity of PD-1/LAG-3 bispecific antibody PD1/LAG3 0927(PD1-LAG3 BsAb) in combination with CD20 CD3 TCB (CD20 TCB) was assessedin vivo in HSC-NSG mice engrafted with the human diffuse large B celllymphoma model WSU-DLCL2, injected s.c. The efficacy of this combinationwas compared to single treatment CD20 CD3 TCB and its combination withNivolumab or Nivolumab plus anti-LAG3 reference antibody.

a) Experimental Material and Methods

Preparation of WSU-DLCL2 cell line: WSU-DLCL2 cells (human diffuse largeB cell lymphoma) were originally obtained from ECACC (EuropeanCollection of Cell Culture) and after expansion deposited in the RocheGlycart internal cell bank. Cells were cultured in RPMI containing 10%FCS and 1× Glutamax. The cells were cultured at 37° C. in awater-saturated atmosphere at 5% CO₂. 1.5×10⁶ cells (in vitro passageP13) per animal were injected s.c. per mouse in RPMI cell culture medium(Gibco) and GFR matrigel (1:1, total volume of 100 ul) at a viability of98.6%.

Production of fully humanized mice: Female NSG mice, in the age of 4-5weeks at the start of the experiment (Jackson Laboratory) weremaintained under specific-pathogen-free condition with daily cycles of12 h light/12 h darkness according to committed guidelines (GV-Solas;Felasa; TierschG). The experimental study protocol was reviewed andapproved by local government (P 2011/128). After arrival, animals weremaintained for one week to get accustomed to the new environment and forobservation. Continuous health monitoring was carried out on a regularbasis. The NSG mice were injected i.p. with 15 mg/kg of Busulfanfollowed one day later by an i.v. injection of 1×10⁵ human hematopoieticstem cells isolated from cord blood. At week 14-16 after stem cellinjection humanized immunodeficient mice (HSC-NSG) were bled sublingualand blood was analyzed by flow cytometry for successful humanization.Efficiently engrafted mice were randomized according to their human Tcell frequencies into the different treatment groups.

Efficacy Experiment: Fully humanized HSC-NSG mice were challengedsubcutaneously with 1.5×10⁶ WSU-DLCL2 cells (human diffuse large B celllymphoma, expressing CD20) at day 0 in the presence of matrigel at 1:1ratio. Tumors were measured 3 times per week during the whole experimentby Caliper. At day 14 (tumor average around 350-400 mm³), the mice wererandomized into seven groups (FIG. 3 ) and the first therapy was given.A weekly scheduled therapy started: Group A received vehicle(phosphate-buffer saline, PBS), Group B received CD20 TCB (0.15 mg/kgonce/week i.v.), Group C received CD20 TCB (0.15 mg/kg once/weeki.v.)+Nivolumab (1.5 mg/kg once/week i.v.), Group D received CD20 TCB(0.15 mg/kg once/week i.v.)+Nivolumab (1.5 mg/kg once/weeki.v.)+anti-LAG3 (BMS-986016, 1.5 mg/kg once/week i.v.), Group E receivedCD20 TCB (0.15 mg/kg once/week i.v.)+PD1-LAG3 BsAb (1.5 mg/kg once/weeki.v.) and Group F received CD20 TCB (0.15 mg/kg once/week i.v.)+PD1-LAG3BsAb (3 mg/kg once/week i.v.). The treatment was given byintra-peritoneal injection in 400 μl max. Tumor growth was measured 3times weekly using a caliper and tumor volume was calculated asfollowed:

T_(v): (W²/2)×L (W: Width, L: Length)

The study was terminated at day 45.

The impact of the therapy was thus assessed by measuring the tumour sizeand displayed as tumour growth over time either as mean (FIG. 4 ), or astumor growth over time for each single mouse (FIGS. 5A to 5F). Forstatistical analysis the last observed tumour volume of each animal wasused as endpoint and it was evaluated if it is below 800 mm³ or not.This endpoint was then subject to pairwise group comparisons based on aChi² test (FIG. 6 ).

b) Results

In this setting, the treatment of WSU-DCLC2 bearing mice with CD20 TCBwas found to mediate strong tumor growth inhibition starting at day 30when compared to the vehicle (FIG. 4 ). As it is known that activationthrough TCB induces PD1 expression as well as LAG3 expression on Tcells, CD20 TCB was combined with either Nivolumab or Nivolumab plus ananti-LAG3 antibody in the attempt to further improve efficacy. However,such combination did not induce a tumour growth reduction to astatistically significant level when compared to the single treatment(FIGS. 4 and 6 ). In contrast, the treatment with PD1-LAG3 BsAb, both at1.5 and 3 mg/kg, in combination with CD20 TCB resulted in a strongtumour protection with strong tumour regression by day 42. When thestatistical analysis was applied considering the last observed tumorsize and fixing the threshold at 800 mm³, a significant increase inanti-tumor efficacy were observed when animals were treated withPD1-LAG3 BsAb at 3 mg/kg in combination with CD20 TCB compared to thetreatment with CD20 TCB in combination with Nivolumab and anti-LAG3antibody.

The tumor growth of each single animal is depicted in the spider plotsin FIGS. 5A to 5F. The plots show that in vehicle all tumors except twoprogressed over the entire experimental window. When CD20-TCB wascombined with Nivolumab or Nivolumab plus anti-LAG3, no majorimprovement of anti-tumor efficacy was observed. In contrast, thecombination of CD20-TCB with PD1-LAG3 BsAb at 3 mg/kg and at 1.5 mg/kgshowed consistent tumor control in most of the mice except one.

Example 5 Potent Anti-Tumor Effect of the Combination Therapy ofPD1/LAG3 Bispecific Antibodies and CD20 CD3 TCB In Vivo in the OCI-Ly18Graft Model in Humanized NSG Mice

In order to evaluate the contribution of PD-1 and LAG-3 co-blockade incombination with CD20 CD3 bispecific antibodies, the combination withCD20 TCB (glofitamab) was assessed in OCI-Ly18 bearing huHSC-NSG mice.The OCI-Ly18 is a human DLBC lymphoma model less sensible to CD20-TCBtreatment which, as monotherapy, failed in controlling tumor growth.

a) Experimental Material and Methods

Production of fully humanized mice: Female NSG mice, age 4-5 weeks atstart of the experiment (Jackson Laboratory) were maintained underspecific-pathogen-free conditions with daily cycles of 12 h light/12 hdarkness according to committed guidelines (GV-Solas; Felasa; TierschG).The experimental study protocol was reviewed and approved by localgovernment (P 2011/128). After arrival, animals were maintained for oneweek to get accustomed to the new environment and for observation.Continuous health monitoring was carried out on a regular basis. The NSGmice were injected i.p. with 15 mg/kg of Busulfan followed one day laterby an i.v. injection of 1×10⁵ human hematopoietic stem cells isolatedfrom cord blood. At week 14-16 after stem cell injection mice were bledsublingual and blood was analyzed by flow cytometry for successfulhumanization. Efficiently engrafted mice were randomized according totheir human T cell frequencies into the different treatment groups.

Preparation of OCI-Ly18 cell line: OCI-Ly18 cells (human diffuse large Bcell lymphoma) were originally obtained from Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH (DSMZ) and after expansiondeposited in the Glycart internal cell bank. OCI-Ly18 cells werecultivated in RPMI 1640 medium (Gibco/Lubioscience #42401-042)containing 10% fetal calf serum (FCS, Gibco) and 1% Glutamax(Invitrogen/Gibco #35050-038). The cells were cultured at 37° C. in awater-saturated atmosphere at 5% CO₂.

Efficacy Experiment: Fully humanized HSC-NSG mice (20 mice per group)were challenged subcutaneously with 5×10⁶ OCI-Ly18 cells (human diffuselarge B cell lymphoma) at day 0 in the presence of matrigel at 1:1ratio. At day 11 (tumor average around 200 mm³) a first treatment withobinutuzumab was administered to eliminate peripheral B cells and avoidcytokine release syndrome. The pre-treatment with obinutuzumab (30mg/kg) was followed by a weekly scheduled therapy of: vehicle (histidinebuffer), CD20 TCB (0.5 mg/kg), PD1-LAG3 BsAb (3 mg/kg), pembrolizumab(1.5 mg/kg) and anti-LAG3 antibody (1.5 mg/kg; antibody comprising theamino acid sequences of SEQ ID NO:79 and SEQ ID NO:80) (i.v.) (see FIG.7 ). Tumor growth was measured 2-3 times weekly using a caliper andtumor volume was calculated as followed:

T_(v): (W²/2)×L (W: Width, L: Length)

The study was terminated on day 35.

b) Results

In this experiment, monotherapy of CD20 TCB (0.5 mg/kg) led to a delayin tumor growth when compared to the vehicle group (FIG. 8 ). However,the combination of CD20 TCB with PD1-LAG3 BsAb (3 mg/kg) provided tumorcontrol and in some mice promoted tumor rejection (FIG. 9C).Interestingly, the combination of CD20 TCB with Pembrolizumab (1.5mg/kg) and anti-LAG3 antibody (1.5 mg/kg) did not differ from CD20 TCBmonotherapy.

These data demonstrate that PD1-LAG3 BsAb improves the anti-tumoractivity of CD20 TCB in the context of lymphoma xenograft models in amanner superior to standard-of-care anti PD-1 antibodies in combinationwith monospecific anti-LAG-3 antibodies administered at 1.5 mg/kg versus3 mg/kg of PD1-LAG3 BsAb in order to match PD-1 and LAG-3 binding sites.These studies establish the contribution of LAG-3 inhibition by PD1-LAG3BsAb over PD-1 inhibition and support its differentiated mechanism ofaction versus competitor anti-PD-1 antibodies in combination withanti-LAG-3 antibodies.

Example 6 Anti-Tumor Effect after Pre-Treatment with Obinutuzumab of theCombination Therapy of PD1/LAG3 Bispecific Antibodies and CD20 CD3 TCBIn Vivo in the OCI-Ly18 Graft Model in Humanized NSG Mice

In this additional experiment, pre-treatment with obinutuzumab, ananti-CD20 depleting antibody, to reduce the cytokine release syndrome(CRS) induced by the peripheral B cell engagement with T cells mediatedby glofitamab was further evaluated (FIG. 10 ).

a) Experimental Material and Methods

Production of fully humanized mice: Female NSG mice, age 4-5 weeks atstart of the experiment (Jackson Laboratory) were maintained underspecific-pathogen-free condition with daily cycles of 12 h light/12 hdarkness according to committed guidelines (GV-Solas; Felasa; TierschG).The experimental study protocol was reviewed and approved by localgovernment (P 2011/128). After arrival, animals were maintained for oneweek to get accustomed to the new environment and for observation.Continuous health monitoring was carried out on a regular basis. The NSGmice were injected i.p. with 15 mg/kg of Busulfan followed one day laterby an i.v. injection of 1×10⁵ human hematopoietic stem cells isolatedfrom cord blood. At week 14-16 after stem cell injection mice were bledsublingual and blood was analyzed by flow cytometry for successfulhumanization. Efficiently engrafted mice were randomized according totheir human T cell frequencies into the different treatment groups.

Preparation of OCI-Ly18 cell line: OCI-Ly18 cells (human diffuse large Bcell lymphoma) were originally obtained from Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH (DSMZ) and after expansiondeposited in the Glycart internal cell bank. OCI-Ly18 cells werecultivated in RPMI 1640 medium (Gibco/Lubioscience #42401-042)containing 10% fetal calf serum (FCS, Gibco) and 1% Glutamax(Invitrogen/Gibco #35050-038). The cells were cultured at 37° C. in awater-saturated atmosphere at 5% CO₂.

Efficacy Experiment: Fully humanized HSC-NSG mice (14 mice per group)were challenged subcutaneously with 5×10⁶ OCI-Ly18 cells (human diffuselarge B cell lymphoma) at day 0 in the presence of matrigel at 1:1ratio. At day 17 (tumor average around 400 mm³) a first treatment withobinutuzumab (30 mg/kg) was administered to eliminate peripheral B cellsand avoid cytokine release syndrome. The obinutuzumab pre-treatment wasfollowed by a weekly scheduled therapy of: vehicle (histidine buffer),CD20 TCB (0.5 mg/kg), PD1-LAG3 BsAb (3 mg/kg), all i.v. (see FIG. 10 ).Tumor growth was measured 2-3 times weekly using a caliper and tumorvolume was calculated as followed:

T_(v): (W²/2)×L (W: Width, L: Length)

The study was terminated on day 35.

b) Results

In this experiment, monotherapy of CD20 TCB (0.5 mg/kg), with apre-treatment with obinutuzumab (30 mg/kg), led to a partial tumorcontrol when compared to the vehicle group (FIG. 11 and FIGS. 12A and12B). However, the combination of CD20 TCB with PD1-LAG3 BsAb (3 mg/kg)provided strong tumor control (FIG. 11 ). In some mice it was observedtumor rejection (FIG. 12C). These data demonstrate that PD1-LAG3 BsAbimproves the anti-tumor activity of CD20 TCB in the context of lymphomaxenograft models also when a pre-treatment with obinutuzumab is used toreduced CRS.

This study established the contribution of PD-1 and LAG-3 inhibition byPD1-LAG3 BsAb over single treatment of CD20-TCB in a context ofobinutuzumab pre-treatment.

1. An anti-CD20/anti-CD3 bispecific antibody for use in a method oftreating CD20-expressing cancer, wherein the anti-CD20/anti-CD3bispecific antibody is used in combination with an anti-PD1/anti-LAG3bispecific antibody, wherein the anti-PD1/anti-LAG3 bispecific antibodycomprises a first antigen binding domain that specifically binds toprogrammed cell death protein 1 (PD1) and a second antigen bindingdomain that specifically binds to Lymphocyte activation gene-3 (LAG3),wherein the first antigen binding domain specifically binding to PD1comprises a VH domain comprising (i) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 1, (ii) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:2, and (iii) HVR-H3 comprising an amino acid sequence ofSEQ ID NO:3; and a VL domain comprising (i) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:4; (ii) HVR-L2 comprising the amino acidsequence of SEQ ID NO: 5, and (iii) HVR-L3 comprising the amino acidsequence of SEQ ID NO:
 6. 2. The anti-CD20/anti-CD3 bispecific antibodyfor use in a method of claim 1, wherein the anti-CD20/anti-CD3bispecific antibody and the anti-PD1/anti-LAG3 bispecific antibody areadministered together in a single composition or administered separatelyin two or more different compositions.
 3. The anti-CD20/anti-CD3bispecific antibody for use in a method of claim 1 or 2, wherein theanti-PD1/anti-LAG3 bispecific antibody comprises a Fc domain that is anIgG Fc domain, particularly an IgG1 Fc domain or an IgG4 Fc domain, andwherein the Fc domain comprises one or more amino acid substitution thatreduces binding to an Fc receptor, in particular towards Fcγ receptor.4. The anti-CD20/anti-CD3 bispecific antibody for use in a method of anyone of claims 1 to 3, wherein the anti-PD1/anti-LAG3 bispecific antibodycomprises a second antigen binding domain that specifically binds toLAG3 comprising (a) a VH domain comprising (i) HVR-H1 comprising theamino acid sequence of SEQ ID NO:11, (ii) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:12, and (iii) HVR-H3 comprising an amino acidsequence of SEQ ID NO:13; and a VL domain comprising (i) HVR-L1comprising the amino acid sequence of SEQ ID NO:14, (ii) HVR-L2comprising the amino acid sequence of SEQ ID NO:15, and (iii) HVR-L3comprising the amino acid sequence of SEQ ID NO: 16; or (b) a VH domaincomprising (i) HVR-H1 comprising the amino acid sequence of SEQ IDNO:19, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:20,and (iii) HVR-H3 comprising an amino acid sequence of SEQ ID NO:21; anda VL domain comprising (i) HVR-L1 comprising the amino acid sequence ofSEQ ID NO:22, (ii) HVR-L2 comprising the amino acid sequence of SEQ IDNO:23, and (iii) HVR-L3 comprising the amino acid sequence of SEQ IDNO:24.
 5. The anti-CD20/anti-CD3 bispecific antibody for use in a methodof any one of claims 1 to 4, wherein the anti-PD1/anti-LAG3 bispecificantibody comprises a first antigen-binding domain specifically bindingto PD1 comprising the VH domain comprising the amino acid sequence ofSEQ ID NO: 9 and the VL domain comprising the amino acid sequence of SEQID NO:
 10. 6. The anti-CD20/anti-CD3 bispecific antibody for use in amethod of any one of claims 1 to 5, wherein the anti-PD1/anti-LAG3bispecific antibody comprises a second antigen-binding domainspecifically binding to LAG3 comprising (a) a VH domain comprising theamino acid sequence of SEQ ID NO: 17 and a VL domain comprising theamino acid sequence of SEQ ID NO: 18, or (b) a VH domain comprising theamino acid sequence of SEQ ID NO: 25 and a VL domain comprising theamino acid sequence of SEQ ID NO:
 26. 7. The anti-CD20/anti-CD3bispecific antibody for use in a method of any one of claims 1 to 3 or5, wherein the anti-PD1/anti-LAG3 bispecific antibody comprises a secondantigen-binding domain specifically binding to LAG3 comprising (a) a VHdomain comprising the amino acid sequence of SEQ ID NO: 27 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 28, or (b) a VHdomain comprising the amino acid sequence of SEQ ID NO: 29 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 30, or (c) a VHdomain comprising the amino acid sequence of SEQ ID NO: 31 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 32, or (d) a VHdomain comprising the amino acid sequence of SEQ ID NO: 33 and a VLdomain comprising the amino acid sequence of SEQ ID NO:
 34. 8. Theanti-CD20/anti-CD3 bispecific antibody for use in a method of any one ofclaims 1 to 6, wherein the anti-PD1/anti-LAG3 bispecific antibodycomprises a first antigen binding domain specifically binding to PD1comprising a VH domain comprising the amino acid sequence of SEQ ID NO:9 and a VL domain comprising the amino acid sequence of SEQ ID NO: 10,and a second antigen binding domain specifically binding to LAG3comprising a VH domain comprising the amino acid sequence of SEQ ID NO:17 and a VL domain comprising the amino acid sequence of SEQ ID NO: 18.9. The anti-CD20/anti-CD3 bispecific antibody for use in a method of anyone of claims 1 to 8, wherein the anti-PD1/anti-LAG3 bispecific antibodycomprises a Fab fragment specifically binding to PD1 and a Fab fragmentspecifically binding to LAG3.
 10. The anti-CD20/anti-CD3 bispecificantibody for use in a method of any one of claims 1 to 6 or 8 or 9,wherein the anti-PD1/anti-LAG3 bispecific antibody comprises a firstheavy chain comprising an amino acid sequence of SEQ ID NO: 35, a firstlight chain comprising an amino acid sequence of SEQ ID NO: 36, a secondheavy chain comprising an amino acid sequence of SEQ ID NO: 37, and asecond light chain comprising an amino acid sequence of SEQ ID NO:38.11. The anti-CD20/anti-CD3 bispecific antibody for use in a method ofany one of claims 1 to 10, wherein the anti-CD20/anti-CD3 bispecificantibody comprises a first antigen binding domain comprising a heavychain variable region (V_(H)CD3) and a light chain variable region(V_(L)CD3), and a second antigen binding domain comprising a heavy chainvariable region (V_(H)CD20) and a light chain variable region(V_(L)CD20).
 12. The anti-CD20/anti-CD3 bispecific antibody for use in amethod of any one of claims 1 to 11, wherein the first antigen bindingdomain comprises a heavy chain variable region (VHCD3) comprising CDR-H1sequence of SEQ ID NO:41, CDR-H2 sequence of SEQ ID NO:42, and CDR-H3sequence of SEQ ID NO:43; and/or a light chain variable region(V_(L)CD3) comprising CDR-L1 sequence of SEQ ID NO:44, CDR-L2 sequenceof SEQ ID NO:45, and CDR-L3 sequence of SEQ ID NO:46.
 13. Theanti-CD20/anti-CD3 bispecific antibody for use in a method of any one ofclaims 1 to 12, wherein a pretreatment with an Type II anti-CD20antibody, preferably obinutuzumab, is performed prior to the combinationtreatment, wherein the period of time between the pretreatment and thecombination treatment is sufficient for the reduction of B-cells in theindividual in response to the Type II anti-CD20 antibody, preferablyobinutuzumab.
 14. A composition comprising an anti-PD1/anti-LAG3bispecific antibody for use in the treatment of CD20 expressing cancer,wherein said treatment comprises administration of said compositioncomprising an anti-PD1/anti-LAG3 bispecific antibody in combination witha composition comprising an anti-CD20/anti-CD3 bispecific antibody,wherein the anti-PD1/anti-LAG3 bispecific antibody comprises a firstantigen binding domain that specifically binds to programmed cell deathprotein 1 (PD1) and a second antigen binding domain that specificallybinds to Lymphocyte activation gene-3 (LAG3), wherein the first antigenbinding domain specifically binding to PD1 comprises a VH domaincomprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, and(iii) HVR-H3 comprising an amino acid sequence of SEQ ID NO:3; and a VLdomain comprising (i) HVR-L1 comprising the amino acid sequence of SEQID NO:4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5,and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:
 6. 15.The composition of claim 13, wherein the anti-PD1/anti-LAG3 bispecificantibody comprises a first antigen-binding domain specifically bindingto PD1 comprising the VH domain comprising the amino acid sequence ofSEQ ID NO: 9 and the VL domain comprising the amino acid sequence of SEQID NO:
 10. 16. The composition of claim 14 or 15, wherein theanti-PD1/anti-LAG3 bispecific antibody comprises a second antigenbinding domain that specifically binds to LAG3 comprising (a) a VHdomain comprising (i) HVR-H1 comprising the amino acid sequence of SEQID NO:11, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:12, and (iii) HVR-H3 comprising an amino acid sequence of SEQ ID NO:13; and a VL domain comprising (i) HVR-L1 comprising the amino acidsequence of SEQ ID NO:14, (ii) HVR-L2 comprising the amino acid sequenceof SEQ ID NO: 15, and (iii) HVR-L3 comprising the amino acid sequence ofSEQ ID NO: 16; or (b) a VH domain comprising (i) HVR-H1 comprising theamino acid sequence of SEQ ID NO:19, (ii) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:20, and (iii) HVR-H3 comprising an amino acidsequence of SEQ ID NO:21; and a VL domain comprising (i) HVR-L1comprising the amino acid sequence of SEQ ID NO:22, (ii) HVR-L2comprising the amino acid sequence of SEQ ID NO:23, and (iii) HVR-L3comprising the amino acid sequence of SEQ ID NO:24.
 17. The compositionof claims 14 to 16, wherein the anti-PD1/anti-LAG3 bispecific antibodycomprises a second antigen-binding domain specifically binding to LAG3comprising (a) a VH domain comprising the amino acid sequence of SEQ IDNO: 17 and a VL domain comprising the amino acid sequence of SEQ ID NO:18, or (b) a VH domain comprising the amino acid sequence of SEQ ID NO:25 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26.18. The composition of claims 14 to 17, wherein the anti-PD1/anti-LAG3bispecific antibody comprises a first Fab fragment specifically bindingto PD1 comprising a VH domain comprising the amino acid sequence of SEQID NO: 9 and a VL domain comprising the amino acid sequence of SEQ IDNO: 10, and a second Fab fragment specifically binding to LAG3comprising a VH domain comprising the amino acid sequence of SEQ ID NO:17 and a VL domain comprising the amino acid sequence of SEQ ID NO: 18.19. The composition of claims 14 to 18, wherein the anti-CD20/anti-CD3bispecific antibody is glofitamab.
 20. The composition of claims 14 to18, wherein the anti-CD20/anti-CD3 bispecific antibody is mosunetuzumab.21. A pharmaceutical composition comprising a combination of ananti-CD20/anti-CD3 bispecific antibody and an anti-PD1/anti-LAG3bispecific antibody for use in the combined, sequential or simultaneous,treatment of a disease, in particular CD20 expressing cancer.
 22. Thepharmaceutical composition of claim 21 for use in the treatment of aCD20 expressing cancer, in particular a hematological cancer selectedfrom the group consisting of Non-Hodgkin lymphoma (NHL), acutelymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuselarge B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle-celllymphoma (MCL), marginal zone lymphoma (MZL), Multiple myeloma (MM) andHodgkin lymphoma (HL).
 23. Use of a combination of an anti-CD20/anti-CD3bispecific antibody and an anti-PD1/anti-LAG3 bispecific antibody in themanufacture of a medicament for treating a CD20 expressing cancer,wherein the anti-PD1/anti-LAG3 bispecific antibody comprises a firstantigen binding domain that specifically binds to programmed cell deathprotein 1 (PD1) and a second antigen binding domain that specificallybinds to Lymphocyte activation gene-3 (LAG3), wherein the first antigenbinding domain specifically binding to PD1 comprises a VH domaincomprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:1, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, and(iii) HVR-H3 comprising an amino acid sequence of SEQ ID NO:3; and a VLdomain comprising (i) HVR-L1 comprising the amino acid sequence of SEQID NO:4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5,and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:
 6. 24.The use according to claim 23, wherein the anti-PD1/anti-LAG3 bispecificantibody comprises a second antigen binding domain that specificallybinds to LAG3 comprising (a) a VH domain comprising (i) HVR-H1comprising the amino acid sequence of SEQ ID NO:11, (ii) HVR-H2comprising the amino acid sequence of SEQ ID NO: 12, and (iii) HVR-H3comprising an amino acid sequence of SEQ ID NO: 13; and a VL domaincomprising (i) HVR-L1 comprising the amino acid sequence of SEQ IDNO:14, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 15,and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16; or(b) a VH domain comprising (i) HVR-H1 comprising the amino acid sequenceof SEQ ID NO:19, (ii) HVR-H2 comprising the amino acid sequence of SEQID NO:20, and (iii) HVR-H3 comprising an amino acid sequence of SEQ IDNO:21; and a VL domain comprising (i) HVR-L1 comprising the amino acidsequence of SEQ ID NO:22, (ii) HVR-L2 comprising the amino acid sequenceof SEQ ID NO:23, and (iii) HVR-L3 comprising the amino acid sequence ofSEQ ID NO:24.
 25. The use according to claims 23 or 24, wherein theanti-PD1/anti-LAG3 bispecific antibody comprises a first Fab fragmentspecifically binding to PD1 comprising a VH domain comprising the aminoacid sequence of SEQ ID NO: 9 and a VL domain comprising the amino acidsequence of SEQ ID NO: 10, and a second Fab fragment specificallybinding to LAG3 comprising a VH domain comprising the amino acidsequence of SEQ ID NO: 17 and a VL domain comprising the amino acidsequence of SEQ ID NO:
 18. 26. A method for treating a CD20 expressingcancer in a subject comprising administering to the subject an effectiveamount of an anti-CD20/anti-CD3 antibody and an effective amount of ananti-PD1/anti-LAG3 bispecific antibody, wherein the anti-PD1/anti-LAG3bispecific antibody comprises a first antigen binding domain thatspecifically binds to programmed cell death protein 1 (PD1) and a secondantigen binding domain that specifically binds to Lymphocyte activationgene-3 (LAG3), wherein the first antigen binding domain specificallybinding to PD1 comprises a VH domain comprising (i) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO: 1, (ii) HVR-H2 comprising theamino acid sequence of SEQ ID NO:2, and (iii) HVR-H3 comprising an aminoacid sequence of SEQ ID NO:3; and a VL domain comprising (i) HVR-L1comprising the amino acid sequence of SEQ ID NO:4; (ii) HVR-L2comprising the amino acid sequence of SEQ ID NO: 5, and (iii) HVR-L3comprising the amino acid sequence of SEQ ID NO:
 6. 27. The method ofclaim 26, wherein the anti-PD1/anti-LAG3 bispecific antibody comprises asecond antigen binding domain that specifically binds to LAG3 comprising(a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequenceof SEQ ID NO:11, (ii) HVR-H2 comprising the amino acid sequence of SEQID NO:12, and (iii) HVR-H3 comprising an amino acid sequence of SEQ IDNO:13; and a VL domain comprising (i) HVR-L1 comprising the amino acidsequence of SEQ ID NO:14, (ii) HVR-L2 comprising the amino acid sequenceof SEQ ID NO:15, and (iii) HVR-L3 comprising the amino acid sequence ofSEQ ID NO:16; or (b) a VH domain comprising (i) HVR-H1 comprising theamino acid sequence of SEQ ID NO:19, (ii) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:20, and (iii) HVR-H3 comprising an amino acidsequence of SEQ ID NO:21; and a VL domain comprising (i) HVR-L1comprising the amino acid sequence of SEQ ID NO:22, (ii) HVR-L2comprising the amino acid sequence of SEQ ID NO:23, and (iii) HVR-L3comprising the amino acid sequence of SEQ ID NO:24.
 28. The method ofclaims 26 or 27, wherein the anti-PD1/anti-LAG3 bispecific antibodycomprises a first Fab fragment specifically binding to PD1 comprising aVH domain comprising the amino acid sequence of SEQ ID NO: 9 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 10, and a secondFab fragment specifically binding to LAG3 comprising a VH domaincomprising the amino acid sequence of SEQ ID NO: 17 and a VL domaincomprising the amino acid sequence of SEQ ID NO:
 18. 29. The method ofany one of claims 26 to 28, the anti-CD20/anti-CD3 bispecific antibodyis glofitamab.
 30. The method of any one of claims 26 to 29, wherein theanti-CD20/anti-CD3 bispecific antibody and the anti-PD1/anti-LAG3bispecific antibody are administered together in a single composition oradministered separately in two or more different compositions.
 31. Themethod of any one of claims 26 to 30, wherein the anti-CD20/anti-CD3bispecific antibody and the anti-PD1/anti-LAG3 bispecific antibody areadministered intravenously or subcutaneously.
 32. The method of any oneof claims 26 to 31, wherein the anti-CD20/anti-CD3 bispecific antibodyis administered concurrently with, prior to, or subsequently to theanti-PD1/anti-LAG3 bispecific antibody.